KaHo Sint-Lieven Campus GILDESTRAAT Gildestraat 17 9000 Gent Belgium
Bachelor in Biomedical Laboratory Technology Option Pharmacy and Biotechnology
Immunohistochemical study of canine
mammary gland tumours
Academical year 2004-2005 Flama Veerle
Uppsala University Rudbeck laboratory Department of Genetics and & Pathology
Dag Hammarskjöldsväg 20 751 85 Uppsala
KaHo Sint-Lieven Campus GILDESTRAAT Gildestraat 17 9000 Gent Belgium
Bachelor in Biomedical Laboratory Technology Option Pharmacy en Biotechnology
Immunohistochemical study of canine
mammary gland tumours
Academical year 2004-2005 Flama Veerle
Uppsala University Rudbeck laboratory Department of Genetics and Pathology
Dag Hammarskjöldsväg 20 751 85 Uppsala
DATA OF THE LABORATORY
Project period: 09/02/2005 – 10/06/2005
Project address: Uppsala University Rudbeck laboratory Department of Genetics and Pathology
Dag Hammarskjöldsväg 20 751 85 Uppsala
Sweden
Project supervisor: Assoc. Prof. Hellmén Eva
ABSTRACT
Immunohistochemical study of canine mammary gland tumours
Headwords: immunohistochemistry, mammary tumours, antibodies
This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies:
- AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells
- CD 31 labelled endothelial cells
- desmin labelled cross-striated and smooth muscle cells
- myosin labelled cross striated muscle cells
- neurofilament (NF) labelled nerve cells
- osteopontin labelled preosteoblasts, osteoblasts and osteocytes
- p63 labelled nuclei of the myoepithelial cells
- smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells
- type I collagen labelled the extracellular matrix in connective tissue and bone
- type II collagen labelled the extracellular matrix in cartilage
- vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells,
nerve cells, macrophages and myoepithelial cells
The tumours were also submitted to a double immunolabelling study using p63 and SMA.
The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings.
Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate.
The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all.
PREFACE
During this project I learned to appreciate the subject immunology and the impact that it has on today’s research. Immunohistochemistry is a mixture of immunology, pathology and histology. This project gave me a clear view and opinion in these matters. But also working with breast tumours made the project even more interesting, because breast cancer is one of the most leading causes of death.
First, I would like to thank my supervisor, Eva Hellmén, for giving me the chance to join her group and to do a part of her research project. Second, I want to thank Helena Wensman, for all the help and support she gave my in the lab.
CONTENTS
DATA OF THE LABORATORY ABSTRACT
PREFACE
1 THE LABORATORY AND THE PROJECT ... 1
2 INTRODUCTION ... 2
3 THEORY AND LITERATURE STUDY ... 3
3.1 ANTIBODIES ... 3 3.1.1 Immunoglobulins ... 3 3.1.2 Polyclonal antibodies ... 4 3.1.3 Monoclonal antibodies... 5 3.1.4 Antibody affinity... 6 3.1.5 Antibody cross-reactivity ... 7 3.2 IMMUNOHISTOCHEMISTRY ... 7
3.2.1 Preparation of tissues for microscopic examination... 7
3.2.2 Antigen retrieval ... 8
3.2.3 Staining methods... 8
3.2.3.1 Direct and indirect staining... 8
3.2.3.2 Two-step indirect method ... 9
3.2.3.3 Vectastain® Elite ABC kit ... 10
3.2.4 Avidin/ Biotin blocking... 11
3.2.5 Basic enzymology ... 11
3.2.5.1 Horse Radish Peroxidase (HRP)... 11
3.3.2.2 Connective tissue ... 17 3.3.2.2.1 Fibers ... 17 3.3.2.2.2 Fibroblasts... 17 3.3.2.2.4 Cartilage... 18 3.3.2.2.5 Bone ... 18 3.3.2.3 Muscle tissue ... 18 3.3.2.3.1 Skeletal muscle ... 18 3.3.2.3.2 Cardiac muscle... 18 3.3.2.3.3 Smooth muscle... 18 3.3.3 Mammary glands ... 19
3.3.3.1 Anatomy of the breast... 19
3.3.3.2 Mammary tumours in the human... 20
3.3.3.2.1 Carcinoma... 20
3.3.3.2.2 Sarcoma ... 20
3.3.3.2.3 Myoepithelial tumours... 21
3.3.3.2.4 Adenomyoepithelioma... 21
3.3.3.3 Mammary tumours in the dog... 21
3.3.3.3.1 Carcinoma... 22
3.3.3.3.2 Sarcoma ... 23
3.3.3.3.3 Mixed mammary gland tumour ... 23
4 MATERIALS AND METHODS ... 24
4.1 ABC – ELITE PROTOCOL WITH DECLOAKING CHAMBER ... 24
4.2 DOUBLE STAINING PROTOCOL ... 25
4.3 ABC-ELITE PROTOCOL WITH Vector® M.O.M.-kit ... 27
4.4 ANTIBODIES IN THE RESEARCH PROJECT... 27
4.4.1 Monoclonal mouse anti-human cytokeratin clones AE1/AE3 ... 29
4.4.2 Monoclonal mouse anti-human vimentin ... 30
4.4.3 Monoclonal mouse anti-human neurofilament ... 31
4.4.4 Monoclonal mouse anti-human type I collagen ... 32
4.4.5 Monoclonal mouse anti-human type II collagen... 33
4.4.6 Monoclonal mouse ascites fluid anti-skeletal myosin ... 34
4.4.7 Monoclonal mouse anti-human desmin ... 35
4.4.8 Monoclonal mouse anti-human smooth muscle actin... 36
4.4.9 Monoclonal mouse anti-human p63 protein... 37
4.4.10 Mouse IgG2a negative control ... 39
4.4.11 Mouse IgG1 negative control ... 39
4.4.12 Polyclonal goat CD 31 PECAM-1 (M-20): sc-1506... 40
4.4.13 Polyclonal Rabbit Anti-Osteopontin ... 41
4.5 TUMOURS... 42
5 RESULTS AND DISCUSSION ... 44
5.1 RESULTS AND DISCUSSION OF THE PRIMARY DOG TUMOURS ... 44
5.2 RESULTS AND DISCUSSION OF THE MOUSE CARCINOMAS ... 45
5.4 RESULTS AND DISCUSSION OF THE DOUBLE STAINING... 51
6 CONCLUSIONS ... 54
1 THE LABORATORY AND THE PROJECT
I performed my project work at the Department of Genetics and Pathology at Uppsala University. The Department is located together with the Department of Oncology, Radiology, Clinical Immunology and the divisions for Clinical Genetics and Pathology-cytology from the Academical Hospital, respectively in the Rudbeck Laboratory. The Department of Genetics and Pathology has some divisions e.g.:
- Medical and clinical genetics - Immunology
- Pathology - Tumour biology - Vascular biology
2 INTRODUCTION
Malignant mammary tumours are one of the most leading causes of death in women. About 1 in 10 women develop breast cancer in their life. 1
It is of great importance to do research on mammary tumours. And just studying the morphology is not good enough. A better knowledge about the phenotype of the tumour is essential and represents a useful tool for predicting the clinical diagnose of the disease.
Little is known about the histogenesis of the special mammary tumours that are investigated in this project. It would be very interesting to know what the origin of the tumours is. Is it epithelial, myoepithelial, mesenchymal?
Immunohistochemistry is the technique that is used to study the tissue sections. They were labelled with different antibodies to obtain important information for the determination of the tumour type. Mostly, monoclonal mouse antibodies were used, such as the antibodies directed against vimentin, desmin, p63, smooth muscle actin, myosin, pancytokeratin AE1/AE3, neurofilament, type I and II collagen. But also the polyclonal goat antibody CD31 and the polyclonal rabbit antibody osteopontin were used.
Examination of certain phenotypes of human mammary tumours is difficult, because they are rare. Breast cancer appears a lot more in dogs than in humans. That is why mammary tumours from a dog were used for the research.
The tumours were surgically removed from the dog, cell lines were made and inoculated subcutaneously into nude mice. When the tumours were fully grown, they were removed and paraffinized, ready to be sectioned and to be examined by different antibodies.
3 THEORY AND LITERATURE STUDY
3.1 ANTIBODIES
The reagent common to all immunohistochemical techniques is the antibody.
3.1.1 Immunoglobulins
Antibodies belong to a group of proteins, called immunoglobulins (Ig). The immunoglobulins comprise five major classes: IgG, IgA, IgM, IgD, IgE. Each immunoglobulin is composed of two identical heavy chains (H) and two identical light chains (L). 3 The structure is shown in figure 1.
Figure 1: The structure of an immunoglobulin 4
The H chains differ in antigenicity and structure and determine the class and subclass of the molecule. The two L chains are either type kappa or lambda. The covalent interchain disulfide bridges join L to H and H to H chains.
The IgG has two monovalent antigen binding fragments (Fab) and one crystalline, constant fragment (Fc). The IgG molecule can be further divided into so-called domains, namely the variable domains and the constant domains (fig.1).
IgM is a pentamer, consisting of five subunits (fig.2). Each subunit is linked by a sulfhydryl-rich peptide, the J chain. It contributes to the integrity and stability of the pentamer.
Figure 2: The structure of an IgM antibody 5
3.1.2 Polyclonal antibodies
Polyclonal antibodies are produced by different cells and are immunochemically dissimilar. They react with various epitopes on the antigen, against which they are raised.
By far, the most frequently used animal for the production of polyclonal antibodies is the rabbit, because of its easy maintenance.
Rabbits weighing approximately 2-3 kg should be chosen. White rabbits are preferable because the node, after injection of bleu dye, is easier to detect through the leg skin. The rabbits should be healthy and adjusted to their new conditions of living.
About a third of the rabbits have endogenous antikeratin antibodies which can give false reaction in immunocytochemistry. This confirms the importance of taking pre-immune serum from a rabbit.
The antigen should be in solution in a sterile and nonimmunogenic solvent such as saline or PBS. About 1 ml of antigen solution will be made into an emulsion with an equal amount of adjuvant, such as Complete or Incomplete Freunds adjuvant. Booster shots, repeated once a month or when decreasing titers are noted, are to maintain or increase antibody levels.
To obtain the antibodies from the immunized rabbit, blood is most harvested from the jugular vein in the ear. The cells are removed from the blood and precipitated by salts, followed by ion exchange chromatography, that serves to remove the bulk of the serum proteins. Affinity chromatography can be used to isolate the antigen - specific antibodies. 3, 6
3.1.3 Monoclonal antibodies
Monoclonal antibodies are the product of an individual clone of plasma cells. Antibodies from a given clone are immunochemically identical and react with a specific epitope on the antigen against which they are raised.
Probably for reasons of economy, mice are currently used almost exclusively for the production. First the mouse is immunized with an appropriate antigen and then cell lines are made, created in vitro by fusing B-lymphocytes, from the spleen, with a myeloma cell line. The fused cell is now called a hybridoma cell.
While B lymphocytes produce the specific antibody, myeloma cells confer immortality in culture medium or in the peritoneal cavity of mice. The hybridoma cells are plated in a special medium (HAT) containing hypoxanthine, aminopterin and thymidine. That allows the growth of some of the hybrides but not the parent cells. Selected hybridomas which are capable of endless reproduction, produce only one kind of antibody that is specific for one kind of antigen. 3, 6
The hybridoma technique is explained in figure 3.
There are certain advantages over their polyclonal counterparts, these include high homogenity, absence of non-specific antibodies, ease of characterization and no batch-to-batch or lot-to-lot variability.
Figure 3: The hybridoma technique that produces monoclonal antibodies 7
3.1.4 Antibody affinity
Ionic interactions, hydrogen bonding and Van der Waals forces are the major contributors to the intrinsic affinity between the paratope of the antibody and the epitope on the antigen. Hydrophobicity appears to have a stabilizing effect on the formed immune complex and may lead to its precipitation. Covalent binding between antibody and antigen does not occur.
The higher the affinity of the antibody, the lower the concentration of free antigen needed for the available binding sites of the antibody to become saturated. Thus high affinity antibodies are desirable and have the advantage that during washing, dissociation is less likely to occur than with low affinity antibodies.
The affinity is also related to their capability to form insoluble immune complexes. The higher the affinity of an antibody, the greater its tendency to form a precipitate.
3.1.5 Antibody cross-reactivity
The term cross-reactivity describes an immunochemical activity that can occur either between an antibody and two or more antigens or vice versa, when an antigen reacts with several different antibodies. It also denotes the interaction of an antibody with similar or dissimilar epitopes on unrelated antigens. Another case is, accidentally induced changes within one or several epitopes, through antigen retrieval, leading to a possible loss of specificity. 3
3.2 IMMUNOHISTOCHEMISTRY
The purpose of histological staining methods is to visualize and differentiate between tissue components, not to determine their chemical composition. On the other hand, histochemical methods show the occurrence and localization of chemical entities in the tissue.
Immunohistochemistry of tissue sections can yield valuable information as to the location of antigens. Thus, it can determine whether the antigen is present in all tissues or only in certain tissues. It can further determine whether all cells in a given tissue are positive for a given antigen, or whether the antigen is restricted to one or a few specialized cell types within the tissue. Its uses are not limited to normal tissues, and testing of tumour tissues can yield information important for determination of tumour type.
Standardisation of a method for staining cytological or histological material requires consideration of all steps in the procedure. 6
3.2.1 Preparation of tissues for microscopic examination
The most common procedure used in the study of tissues is the preparation of histologic sections. Under the light microscope, tissues are examined by transillumination, so sections must be thin and translucent.
Before preparing the sections, the tissue should be adequately treated with chemical substances, called fixatives. This step is to avoid tissue digestion by lysosomal enzymes (autolysis), bacteria or moulds and to preserve the physical structure. Further more the fixatives stabilize the cells and tissues to protect them from the rigors of subsequent processing and staining techniques. 6
There are different kinds of fixatives, only the fixation in formalin, that is used in this project, will be further discussed.
Neutral buffered formalin solution (NBF) 10%
Formalin (40% w/v formaldehyde) 100 ml
Sodium phosphate, monobasic, monohydrate 4 g (c = 0,0290 mol/l) (NaH2PO4.H2O M.W.=138,0 g/mol)
Sodium phosphate, dibasic, anhydrous 6,5 g (c = 0,0458 mol/l) (Na2HPO4 M.W.=141,96 g/mol)
This solution is stable for many months at room temperature. Small blocks of tissue should be fixed for up to 24 hours. 3
Tissues must be infiltrated after fixation with embedding substances that give a rigid consistency to the tissue, which is necessary to obtain thin sections. Paraffin is used routinely for light microscopy. In general, more antibodies will react on frozen sections than on paraffin sections, however, morphology is better preserved in the paraffin embedded sections.
The sections are precisely cut by fine cutting instruments called microtomes. In this project, the small paraffin tissue blocks are sectioned by the microtomes’ steel blade to a thickness of 5 µm. The sections are floated on warm water and transferred to glass slides. 6
3.2.2 Antigen retrieval
The loss of immunoreactivity by many antigens caused as a result of fixation in formalin has introduced many challenges.
Although formalin fixation will allow some epitopes to emerge unchanged, others will undergo substantial changes. In this process, cross-linking of unrelated proteins to the target antigen is also possible. To improve the immunoreactivity, proteolytic enzymes can be used: bromelain, chymotrypsin, ficin, pepsin, pronase and various other proteases. The enzyme compensates for the impermeable nature of the non-coagulant fixatives by etching the tissue and allowing hidden determinants to be exposed. Use of enzymes may however also entail the risk of destroying some epitopes. But still for some antibodies it is better to use an enzyme.
Another approach for the restoration of immunoreactivity is the use of solutions containing various metals and microwave heating. The immunoreactivity is recovered through exposure of heat near the boiling point of water. After deparaffinizing and dehydrating, the tissue sections are immersed in an aqueous solution commonly referred to as a ‘retrieval solution’. The container that holds the slides is exposed to heat in the decloaking chamber. 3, 8
If false positive staining and non-specific background staining occur, then a new technique can be used, the combination of mild conditions of heat-treatment and enzyme digestion instead of maximal heating condition. 9
3.2.3 Staining methods
3.2.3.1 Direct and indirect staining
There are both direct and indirect methods for antigen localization.
The indirect method is a more sensitive technique. Antibodies to an antigen are produced in an animal, for example in a rabbit. Rabbit immunoglobulins are, in turn, capable of inducing antibody response in other animals such as goat, thus producing an anti-antibody. A tissue section containing the antigen is incubated with unlabeled rabbit antibodies. After washing, labelled goat anti-rabbit antibodies are added. 6, 8
3.2.3.2 Two-step indirect method
In the research project, the two-step indirect method is used. First an unconjugated primary antibody binds to the antigen. An enzyme-labelled secondary antibody directed against the primary antibody is then applied, followed by the substrate-chromogen solution. If the primary antibody is made in rabbit or mouse, the secondary antibody must be directed against rabbit or mouse immunoglobulins, respectively.
This method is more versatile than the direct method, because a variety of primary antibodies from the same species can be used with the same conjugated secondary antibody. The procedure is also more sensitive, as several secondary antibodies are likely to react with a number of different epitopes of the primary antibody.
Undesired reactions may occur if the secondary antibody cross-reacts with immunoglobulins of the tissue specimen. This is now routinely eliminated by using pre-absorbed secondary antiserum, that has been absorbed with immunoglobulins from the species under investigation.
Most of the immunochemical staining methods in use today are based on the high affinity that Streptavidin (Streptomyces avidinii) and avidin (chicken eggs) have for biotin. Both posses four binding sites for biotin, but due to the molecular orientation of the binding sites, fewer than four molecules of biotin will actually bind.
The method consists of primary antibody, biotinylated secondary antibody followed by, in this study, the avidin-biotin-complex (ABC) technique and concludes with the substrate solution. Horse radish peroxidase is used as enzyme label. 3, 6
Figure 4: The ABC-technique 10
3.2.3.3 Vectastain® Elite ABC kit
The Vectastain® Elite ABC kit (Avidin and Biotinylated horse radish peroxidase macromolecular Complex) is one of the most sensitive, economical and reliable immunoperoxidase systems available. This enhanced sensitivity is particularly important in the localization of antigens present in low amounts or in cases where the cost of primary antibodies is significant. The increased sensitivity also provides an option to substantially reduce staining times.
This technique employs unlabelled primary antibody, followed by biotinylated horse radish peroxidase macromolecular complex. This has been termed the ABC technique.
Formation of the complex is achieved by mixing avidin and biotinylated horse radish peroxidase in dilute solution and in defined amounts prior to use.11
The Vectastain® working solutions are:
- blocking serum: the preferred serum for blocking is prepared from the same species in which the biotinylated secondary antibody is made.
- Biotinylated secondary antibody - Vectastain® Elite ABC reagent
3.2.4 Avidin/ Biotin blocking
The blocking kit reagents are used to block nonspecific binding of biotin/avidin system reagents. Some tissues may bind avidin, biotinylated horse radish peroxidase or other biotin/avidin system components without prior addition of biotinylated antibody. This binding may be due to endogenous biotin or biotin-binding proteins, lectins, or nonspecific binding substances present in the section. If a high background is present using the ABC reagents in the absence of biotinylated secondary antibody, pre- treatment of the tissue with avidin, followed by biotin, to block the remaining biotin binding sites on the avidin, may be required. 12
3.2.5 Basic enzymology
Immunoenzymatic staining methods utilize enzyme substrate reactions to convert colourless chromogens into coloured end products. A very broad classification of enzymes would include hydrolytic enzymes (esterases, proteases), phosphorylases, oxidoreductive enzymes (dehydrogenases, oxidases, peroxidases), transferring enzymes, decarboxylases and others. Enzymatic activity is dependent upon several variables such as enzyme and substrate concentrations, pH, salt concentration of the buffer milieu, temperature and light. Horse Radish Peroxidase will be considered in detail. 3
3.2.5.1 Horse Radish Peroxidase (HRP)
3.2.5.2 Substrate
The Vector® NovaRED is used as enzyme/substrate for all the immunostainings. It produces a red reaction product.
For the double staining protocol, different substrates are used: Vector® NovaRED, Vector® SG that yields a grey stain and Vector® DAB that stains the tissue sections brownish. 13, 14, 15
3.2.6 Mayer’s hematoxylin
After the addition of substrate, the sections are counterstained with Mayer’s Hematoxylin. It behaves like a basic dye, it labels the basophilic tissue components (nucleoproteins, glycosaminoglycans, and acid glycoproteins). Hematoxylin stains the cell nucleus and other acidic structures, such as RNA-rich portions of the cytoplasm, blue. 6
Before the mounting of the sections, they are washed, and dehydrated in 2x 95% ethanol, 2x 100% ethanol and xylene.
3.2.7 Controls
Reagent and tissue controls are necessary for the validation of immunohistochemical staining results. Without their use, interpretation of staining would be of doubtful value.
The relevant reagents must be controlled within routinely conducted quality assurance programs both by manufacturer as well as by the user. The prime objective is to ascertain whether the primary and secondary antibodies are specific for their targets.
Tissue controls can be of either the negative, positive or the internal type. Negative tissue controls do not contain the relevant tissue marker.
3.3 HISTOLOGY
3.3.1 Cell components
The cell is the functional unit of all living tissues. Each cell has the capacity to perform all the essential life functions.
The cell is composed of two basic parts : cytoplasm and nucleus. The outermost component of the cell is the plasma membrane. The cytoplasm itself is composed of a matrix in which are embedded several organelles, also deposits of carbohydrates, lipids and pigments (see figure 5). 8,
16
Figure 5: The composition of a human cell 17
3.3.1.1 Cell nucleus
3.3.1.2 Cytoplasm
3.3.1.2.1 Cytoskeleton
The cytoplasmic matrix contains a complex network of microtubules, microfilaments and intermediate filaments. These structural proteins not only provide for the form and shaping, but also play a role in cytoplasmic and cellular movement. Only the intermediate filaments will be discussed in detail.
Intermediate filaments have a stable fibrous structure specific to particular cell types.
- Cytokeratins are found in most epithelia and constitute a family of approximately 20
polypeptides.
- Vimentin filaments are characteristic of cells of mesenchymal origin (fibroblasts) and of
embryonic or undifferentiated cells. Vimentin is a single protein and may copolymerize with desmin or glial fibrillary acidic protein.
- Desmin is found in smooth muscle and in the Z-disks of skeletal and cardiac muscle.
- Glial filaments are characteristic of astrocytes but are not found in neurons, muscle,
mesenchymal cells or epithelia.
- Neurofilaments consist of at least three high-molecular-weight polypeptides. They have different chemical structures and different roles in cellular function. They are expressed in nerve cells (neurons).
The presence of a specific type of intermediate filament in tumours is an important characteristic that can affect not only the diagnosis but also the treatment of tumours. Identification of intermediate filament protein is a routine procedure using immunohistochemical methods. 8, 16
3.3.1.2.2 Plasma membrane
All eukaryotic cells are enveloped by limiting membrane composed phospholipids and cholesterol, proteins and oligosaccharides covalently linked to some of the lipids and proteins. The plasma membrane functions as a selective barrier that regulates the passage of certain materials into and out of the cell. It plays an important role in the way a cell interacts with its environment. 8, 16
3.3.1.2.3 Organelles
The different organelles of the cytoplasm are described. 8, 16
Mitochondria
Ribosomes
Ribosomes are small–electron-dense particles about 20x30 nm in size. They are composed of four types of ribosomal RNA and almost 80 different proteins. In eukaryotic cells, the RNA’s of both subunits are synthesized in the nucleoli of the nucleus. Their proteins are synthesized in the cytoplasm and then enter the nucleus and associate with rRNA. Subunits then leave the nucleus, via nuclear pores, to enter the cytoplasm and participate in protein synthesis.
Endoplasmic reticulum
Endoplasmic reticulum is the site of lipid and carbohydrate synthesis, protein segregation from the cytoplasm, and the initial posttranslational modifications that prepare proteins for their specific function. There are two specialized types of endoplasmic reticulum, namely rough an smooth.
Golgi complex
It completes posttranslational modifications, packages and places an address on products that have been synthesized by the cell.
Lysosomes
Lysosomes are sites of intracellular digestion. They contain a large variety of hydrolic enzymes. Lysosomal enzymes are synthesized and segregated in the rough endoplasmic reticulum and transferred to the Golgi complex where they are modified and packaged as lysosomes. They can digest materials taken into the cell from its environment, a process called heterophagy.
Secretory granules
Secretory granules are found in those cells that store a product until its release is signalled by a metabolic, hormonal or neural message.
3.3.2 Basic histology
The human body is composed of 4 basic types of tissue: epithelial, connective, muscular and nervous. Each of these tissues is composed of several cell types. Approximately 200 types of cells are recognized in the human body.
3.3.2.1 Epithelial tissue
The epithelial tissue is composed of polyhedral cells with very little intercellular substance. The principle functions of epithelial tissues are the covering and lining of surfaces, absorption, secretion, sensation and contractility (myoepithelium). Epithelia are derived from all three embryonic germ layers: ectoderm, endoderm and mesoderm.
The forms and dimensions are varied, ranging from high columnar to cuboidal to low squamous cells.
All epithelial cells, in contact with subjacent connective tissue, present at their basal surface a sheet-like extracellular structure called basal lamina. 8
3.3.2.1.1 Glandular epithelia
Glandular epithelia is formed by cells specialized to produce a fluid secretion that differs from blood or intracellular fluid. The mammary glands secrete proteins, lipids and carbohydrates. 8
3.3.2.1.2 Lamina propria
The lamina propria is a layer of connective tissue which is bound to the epithelium by the basal lamina. It supports the epithelium and binds it to neighbouring structures. 8
3.3.2.1.3 Myoepithelial cells
The myoepithelial cells embrace gland acini. They are longitudinally arranged along ducts in two layers, the luminal layer and the basal myoepithelial layer. The whole structure is surrounded by a basement membrane. The myoepithelial layer is organised differently in the ducts and in the lobules.
The myoepithelial cells contain numerous actin microfilaments, as well as tropomyosin and myosin. They also contain intermediate filaments that belong to the cytokeratin family, dense plaques, smooth muscle and specific cytoskeletal and contractile proteins.
Their function is to contract around the secretory or conducting portion of the gland and thus help to propel secretory products towards the exterior.
3.3.2.2 Connective tissue
The connective tissue provides a matrix that serves to connect and bind the cells and organs and give support to the body. The major constituent is its extracellular matrix composed of protein fibers and amorphous ground substance and tissue fluid. Embedded in the extracellular matrix, are the connective tissue cells.
The connective tissue can be subdivided into three classes of components: cells, fibers and ground substance.
The connective tissue serves as the medium through which nutrients and metabolic wastes are exchanged between cells and their blood supply.
Phagocytic cells engulf inert particles and micro-organisms that enter the body. Antibodies are produced by plasma cells in the connective tissue. 8
3.3.2.2.1 Fibers
Connective tissue fibers are long, slender protein polymers that are present in variable proportions in the different types of connective tissue. There are 3 main types of connective tissue fibers: collagen, reticular, elastic. Collagen and reticular fibers are formed by the protein collagen, and the elastic fibers are composed of the protein elastin.
There are different types of collagen fibers. Collagen type I and II are important because, in the project antibodies against these types are used.
Collagen type I is the most abundant and distributed. It occurs in collagen fibers that form bones, dentin, dermis, etc. The fibers can form bundles. Collagen type II is mainly present in hyaline and elastic cartilage. Only very thin fibrils are formed, and no fibers. 8
3.3.2.2.2 Fibroblasts
Fibroblasts are the cells most commonly found in connective tissue. They are responsible for the synthesis of fibers and amorphous intercellular substance. 8
Most connective tissues develop from the mesoderm, that is the middle layer of the embryo. Mesodermal cells migrate from their site of origin, surrounding and penetrating developing organs. These are the mesenchymal cells, also called the fibroblasts. They are characterized by an oval nucleus with prominent nucleoli and fine chromatin. They have an irregular, star shape with branching cytoplasmic extensions which form a network. 16
3.3.2.2.3 Adipose tissue
The adipose tissue is a special kind of connective tissue in which adipocytes predominate. It is one of the largest organs in the body. 8
3.3.2.2.4 Cartilage
Cartilage consists of chondrocytes and an extensive extracellular matrix composed of fibers and ground substance. The cells are located in matrix cavities, called lacunae. Three forms of cartilage have evolved: hyaline cartilage, elastic cartilage and fibrocartilage. 8
3.3.2.2.5 Bone
Bone is a specialized connective tissue composed of bone matrix, osteocytes in cavities, osteoblasts and osteoclasts. 8
3.3.2.3 Muscle tissue
There are three types of muscle cells.
3.3.2.3.1 Skeletal muscle
Skeletal muscle is composed of bundles of very long, cylindrical, multinucleated cells that show cross-striations. Their contraction is quick, forceful, and under voluntary control. It is caused by the interaction of thin actin filaments and thick myosin filaments. 8
3.3.2.3.2 Cardiac muscle
The cardiac muscle also has cross-striation and is composed of elongated, individual cells that lie parallel to each other. The contraction is involuntary, vigorous and rhythmic. 8
3.3.2.3.3 Smooth muscle
The smooth muscle consists of collections of fusiform cells that do not show cross-striation. Their contraction is slow and not subject to voluntary control. Each cell has a single nucleus in the center of the cell.
3.3.3 Mammary glands
3.3.3.1 Anatomy of the breast
The breast is a mass of glandular, fatty and fibrous tissues, positioned over the pectoral muscles of the chest wall. The breast is composed of:
-milk glands (lobules) that produce milk
-ducts that transport milk from the lobules to the nipple -nipple
-areola (brown pigmented region surrounding the nipple) -connective (fibrous) tissue that surrounds the lobules and ducts -adipose tissue
Each mammary gland is a highly modified apocrine sweat gland that consists of 15-25 lobules whose function is to secrete milk to nourish newborns. Each lobule, separated from the others by dense connective tissue and much adipose tissue, is a gland in itself with its own excretory lactiferous duct. These ducts, emerge independently in the nipple, which has 15-25 openings (figure 6).18
The lining of the lactiferous ducts and terminal interlobular ducts is formed by simple cuboidal epithelium with oval nuclei and covered by closely packed myoepithelial cells.18
In the project, a carcinoma and an osteosarcoma were examined. Only these two kinds of tumours will be further discussed. First, they are described in the human and then the classification in the dog is explained.
3.3.3.2 Mammary tumours in the human
Just like all other cells in the human body, breast cellsmultiply. The increase of cells is normally regulated by diverse control mechanisms. But sometimes, these mechanisms fail, and lead to an abnormal growth of the tissue, that can expand to the surrounding tissues and further areas. 20
Non-invasive tumours stay within the milk ducts or milk lobules in the breast. They do not grow into or invade normal tissues within or beyond the breast. These can be called ‘in situ’ or precancers. If the tumour has grown beyond where it started, it is called invasive and most tumours are. 21
Invasive breast tumours are composed of proliferated tumour cells derived from the mammary ductal or arcinar epithelium or of a host stromal. Stromal cells are composed of fibroblasts and fibroblast-like cells such as myofibroblasts, but also of macrophages and blood capillaries.
The reactive stroma is essential for tumour growth and may actively participate in tumour invasion and metastasis through tissue remodelling. 22
3.3.3.2.1 Carcinoma
Most of the cancers arise from epithelial cells of the lactiferous ducts, they are called carcinomas. If these cells metastasize to the lungs, brain, or bone, breast carcinoma becomes a major cause of death. 20
The histologic classification of breast carcinomas includes a variety of tumour types. The majority belongs to the category of invasive ductal carcinomas of no specific type. But almost 30 % of invasive breast carcinomas, however are of a special type and most of them carry specific prognostic implications. 21
3.3.3.2.2 Sarcoma
A sarcoma is a malignant tumour of mesenchymal tissue that destroys the affected organs very fast. Mostly, metastases appear in other parts of the body. 23
Malignant phyllodes tumour has epithelial elements within a cellular stroma that had undergone malignant change. Stromal sarcomas, in contrast, lack the epithelial component and are formed entirely from within the breast tissue.
The cause of breast sarcoma is unknown. It is suggested that a fibroadenoma may be a precursor of the sarcoma. However, fibroadenomas have traditionally been regarded as benign breast tumours.
Breast sarcoma usually is a painless, mobile and large breast mass. There is no nipple discharge, skin or nipple involvement, or axillary adenopathy. The clinical features tend to be those of a typical fibroadenoma. However, a history of rapid growth of an indolent lump should raise the suspicion of a sarcoma. It should also be considered in older patients. 24
3.3.3.2.3 Myoepithelial tumours
Most mammary myoepithelial tumours are benign, and typically show tubules with inner epithelial ductal cells enclosed by clear proliferating myoepithelial cells.
Myoepithelial cells are capable of forming various kinds of tumours in the gland. Although malignant myoepithelial tumours of the breast are rare, the study of their peculiar morphology as well as the histochemical and ultrastructural features will uncover more cases. 25
Several members of the epidermal growth factor family have been shown to play an important regulatory role in mammary gland development and in the differentiation of mammary epithelium. It is suggested that the epidermal growth factor receptor plays a specific role in signalling in the control of the basal myoepithelial cell phenotype.
The most recently discovered members of the p53 family are p63 and p73. These transcription factors play a central role in the control of cell growth and survival, and they act as tumour suppressors.
The analysis of myoepithelial marker expression remains a commonly used approach to distinguish between benign and malignant tumours, or to detect stromal invasion. 3
3.3.3.2.4 Adenomyoepithelioma
Adenomyoepithelioma is a prominent proliferation of both epithelial and luminal and peripherical myoepithelial cells and has been considered to be a benign lesion, although local recurrences have been described especially in the tubular type. The histogenesis is unclear. 25, 26
3.3.3.3 Mammary tumours in the dog
Particularly the study of malignancies has received much attention, since breast carcinomas with different keratin compositions may exhibit different clinical behaviours, and the vimentin expression of these tumours has been associated with progressive behaviour. 27
3.3.3.3.1 Carcinoma
The carcinomas are classified in four groups. 28
Noninfiltrating (in situ) carcinoma is an epithelial neoplasm with histologic features of
malignancy that has not invaded through the basement membrane.
Complex carcinoma is composed of both luminal epithelial and myoepithelial components. The
myoepithelliumlike cells are often of spindle cell type. Expansive, lobulated growth is quite common, and growth within lymphatics is about 10%.
Simple carcinoma is composed of one type of cell either luminal epithelial cells or myoepithelial
cells. These tumours have a strong tendency to infiltrate into surrounding tissues and vessels. Lymphatic and hematogenous spread is common.
The simple carcinomas can be further divided into three classes. The first class is the tubulopapillary carcinoma, it is characterized by the formation of tubules and/or papillary projections. Second, the solid carcinoma, this one is characterized by the arrangement of tumour cells in solid sheets, cords or nests. And at last the anaplastic carcinoma, it is an highly infiltrative tumour of pleomorphic epithelial cells that is not classifiable in one of the other categories of carcinoma.
Special carcinomas:
- Spindle cell carcinoma or myoepithelioma is a malignant tumour composed of spindle cells that are usually arranged in epithelial patterns. Some are solid, while others may also contain some tubules. Differentiation between spindle cell carcinoma and fibrosarcoma can be done by using the immunohistochemical markers cytokeratin and vimentin. It seems likely that some spindle cell carcinomas are of myoepithelial origin. The tumour is relatively rare in the dog.
- Squamous cell carcinoma is composed of solid sheets and cords of cells with areas of squamous differentiation. Most of these tumours are of a highly infiltrative type, and invasion of lymphatics is common.
- Mucinous carcinoma is characterized by abundant mucin production.
- A matrix-producing breast carcinoma is an extremely rare type of metaplastic carcinoma. High-grade epithelial component continually merges with heterologous mesenchymal chondroid component without clearly spindle cell sarcomatoid pattern in between.
The heterologous mesenchymal chondroid component can be produced in two different ways, one displayed typical structure of low-grade hyaline cartilage, in the second one, the epithelial tumourous cells were embedded in the homogenous eosinophillic extracellular matrix giving appearance of chondroid aura.
It differs from conventional myoepithelial carcinoma and from heterologous metaplastic carcinoma. It is a very aggressive lesion. 29
3.3.3.3.2 Sarcoma
The dog sarcomas are classified in two groups 28 :
- Fibrosarcoma is a malignant tumour of fibroblasts with variable amounts of collagen.
The tumours are composed of spindle cells that have formed reticulin and collagenous fibers.
- Extraskeletal osteosarcoma is a rare mesenchymal tumour that is characterized by
osteoid and/or bone formation by neoplastic cells, without primary periosteal or bone involvement. Combined tumours are composed of malignant osseous and cartilaginous tissue. The malignant fibrous tissue and/or adipose tissue can be present.
The aggressive osteosarcoma is associated with early hematogenous metastasis, frequently to the lung, and a short survival time. 30, 31
3.3.3.3.3 Mixed mammary gland tumour
4 MATERIALS AND METHODS
Immunohistochemistry was performed on different tumour sections. The tumours were derived from cell lines, derived from mammary dog tumours. These were subcutaneously injected in nude mice. Once the tumours were fully grown in the mice, they were surgically removed, fixed in 10 % buffered formalin and embedded in paraffin. Sections with a thickness of 5 µm were cut with the microtome and kept at + 37 °C over night. Then, they were stored in the refrigerator room at – 10 °C.
4.1 ABC – ELITE PROTOCOL WITH DECLOAKING CHAMBER
The tumour sections were deparaffinized and rehydrated through xylene and graded alcohol series. Then, they were boiled in the decloaking chamber ( Biocare Medical). The outer chamber was filled with 500 ml tap water. The plastic jar with the sections was filled with diluted Antigen unmasking solution ( Vector® 1:100). The lid was put on and the pressure program was started. After the boiling step, the sections were left in the chamber for one hour with the heat on.
The sections were rinsed, first in distilled water and then in Tris buffer (0,05 mol/l Tris-HCl pH 7,6). After the rinsing, the sections were incubated with respectively, avidin and biotin (50 µl per section) for 30 minutes at room temperature. After each incubation they were rinsed in Tris buffer. The sections were then incubated in blocking serum (e.g. Normal Horse Serum 1:50) for 60 minutes. The excess of the serum was blotted from the sections and the primary antibody, diluted in Tris buffer, was put on the sections and then placed in the refrigerator room at + 4 °C over night.
After rinsing in Tris buffer, with the negative controls in a separate jar, the sections are incubated with biotinylated secondary antibody diluted in Tris buffer for 30 minutes at room temperature. Biotinylated mouse (1:200) was used for monoclonal mouse antibodies and biotinylated anti-rabbit (1:200) for polyclonal antibodies. Meanwhile the ABC-Elite reagent (20 μl A + 20 μl B in 1 ml Tris buffer) was mixed and kept at room temperature for 30 minutes before use. The sections were rinsed again with Tris buffer and incubated with ABC reagent for 45 min at room temperature. After rinsing in Tris buffer, the substrate, Vector® Nova RED, was put on the sections. The development time was determined under the light microscope. The sections were washed for 5 minutes in tap water
Counterstaining was done with Mayer’s hematoxylin for 2 or 3 minutes. After rinsing with tap water, the sections were rinsed two times in 95% ethanol and in 100% ethanol. Then the sections were mounted in Pertex.
Preparations:
0,5 mol/l Tris-HCl buffered in 9% NaCl pH 7,6
302,85 g Tris 450,00 g NaCl
Dissolve the amount in 4500 ml distilled water. Adjust the pH with approximately 240-245 ml 25% HCl to pH 7,6. Fill up to 5000 ml with distilled water.
Dilute the stock solution x 10 upon use: 100 ml + 900 ml distilled water = 0,05 mol/l Tris-HCl.
Pepsin solution
Pour 10 ml milli-Q H2O in a 50 ml bottle. Milli-Q water is distilled water that is feed through a
special ion exchange cartridge to increase the purity. Add 13 µl of 25 % HCl. Then, add 0,387 g pepsin to the solution and dissolve it. The pepsin solution should be stored in the refrigerator.
Substrate Vector® Nova Red
Immediately before use on tissue sections, the substrate solution should be prepared.
Fill a tube with 5 ml distilled water, add three drops of reagent 1 and mix well, then add two drops of reagent 2, 3 and hydrogen peroxide to the solution and mix well. All the products needed for the preparing of the substrate are available in the Vector® kit.
Incubate tissue sections with the substrate at room temperature until suitable staining develops. Development times should be determined by the investigator under the light microscope. Then the sections are rinsed in tap water.
Little is known about the toxity and carcinogenicity of the substrate kit components. Care should be exercised when using these reagents including gloves, eye protection, lab coats, and good laboratory procedures. Dispose the used working solution with care.13
4.2 DOUBLE STAINING PROTOCOL
The labelling of multiple antigens in the same tissue section was performed with Vectastain® ABC kits.
First, the sections were deparaffinized and rehydrated in xylene and graded alcohol series. After they were rinsed in tap water, the antigen retrieval method was performed. After rinsing with distilled water and Tris buffer, the sections were incubated with respectively avidin and biotin for 15 minutes (50 µl per section). In between and afterwards, were the sections rinsed with Tris buffer.
At that moment, the ABC-Elite reagent from the first kit was also prepared (20µA + 20 µl B + 1 ml Tris buffer). The sections were rinsed and incubated with ABC-Elite reagent (50 µl per section) for 30 minutes.
After this the first substrate was prepared and added on the sections and after developing time, rinsed with Tris buffer. Now, the first antigen was labelled.
To label the second antigen, the same procedure was followed, starting from the blocking step with the NS. Except now, the second kit was used. But if the two antibodies are both monoclonal or polyclonal, the same kit can be used further.
The protocol was followed by a counterstaining, if that was desired. Then, the sections were dehydrated, twice in 95 % and in 100 % ethanol. And finally the sections were mounted with Pertex.
In this study, different enzyme/substrate combinations with the substrates Vector® Nova RED, Vector® SG (grey) and Vector® DAB (brown) were performed. Also, the Vector® methyl green nuclear counterstain was tested on the sections.
Preparations:
Substrate Vector® SG
To 5 ml of Tris buffer add 3 drops of chromogen and mix well. Then, add 3 drops of the hydrogen peroxide solution and mix well. Incubate the substrate at room temperature. The development times should be determined under the microscope by the investigator. Then, the sections are washed in Tris buffer solution for 5 minutes and then rinsed briefly in water.14
Vector® methyl green nuclear counterstain
First, rinse the slides in tap water. Then preheat the Vector® methyl green solution to 60°C in a jar. Add the slides and incubate them at 60°C for 1-5 minutes. After incubation, remove the slides and rinse with distilled water until the rinse water is clear. Wash the slides for 1 minute in distilled water. Next, dip the slides 5-10 times in acetone containing 0,05 % (V/V) acetic acid. Immediately, dehydrate, clear and permanently mount the slides.
Use ordinary precautions to avoid contact with skin and eyes. Methyl green stains the nuclei of the cells light green. 32
Substrate Vector® DAB (3,3’-diaminobenzidine)
To 5 ml of distilled water, add two drops of Buffer Stock Solution and mix well. Then, add four drops of DAB Stock Solution and two drops of Hydrogen Peroxide Solution and mix well. Incubate tissue sections with the substrate at room temperature and evaluate the staining development under light microscope. Wash the sections for five minutes in water.
4.3 ABC-ELITE PROTOCOL WITH Vector® M.O.M.-kit
In some cases when mouse monoclonal antibodies were used on mouse tissue, a very high background was present. That made the interpretation of the tumours very difficult, because the background obscured the specific staining.
The major problem is the inability of the anti-mouse secondary antibody to distinguish between the mouse primary antibody and the endogenous mouse immunoglobulins in the tissue.
Vector® created a special kit, M.O.M. (mouse on mouse), to eliminate the background problem. First, the tumour sections were deparaffinized and hydrated through xylene and graded alcohol series. The sections were rinsed for 5 minutes in tap water and then incubated with antigen unmasking solution. The method of antigen retrieval depends on the used primary antibody and the tissue.
Then, the sections were rinsed in Tris buffer and incubated with respectively, avidin and biotin (50 µl per section), with a Tris buffer rinsing step in between en afterwards. In the next step the sections were incubated for 60 minutes with M.O.M. working solution mouse Ig blocking reagent.
After the rinsing in Tris buffer, M.O.M. working solution diluent (50 µl per section) was put on the tissue sections for 5 minutes. Then, the excess was blotted and the primary antibodies were diluted in M.O.M. working solution diluent to obtain the right concentration. They were put on the tissue sections (50 µl per section) and followed by an overnight incubation at 4°C.
The second day, the sections were rinsed in Tris buffer and the negative controls were placed in a separate jar. The sections were incubated with M.O.M. working solution biotinylated anti-mouse Ig reagent (50 µl per section) for 10 minutes. Meanwhile the ABC-Elite reagent was prepared (20 μl A + 20 μl B in 1 ml Tris buffer) and kept at room temperature for 30 minutes before use. After the rinsing with Tris buffer, the sections were incubated with ABC reagent for 45 min at room temperature. At last, the substrate/enzyme was added and the sections were counterstained in Mayer’s hematoxylin for 2 or 3 minutes. After rinsing with tap water, the sections were rinsed two times in 95% ethanol and in 100% ethanol, followed by the mounting in Pertex. 33
4.4 ANTIBODIES IN THE RESEARCH PROJECT
A preliminary study on normal tissue was performed to optimize the immunostaining conditions for all the selected antibodies. First, each of the antibodies was tested strictly in accordance with the manufacturer’s recommended ABC-Elite protocol. Then, the dilutions were adjusted until the best stainings were obtained.
Table 1: The negative and positive control, working dilution and pretreatment of the antibodies
Antibody Negative control Positive control Dilution Pre-treatment
AE1/AE3 (pancytokeratin) IgG1 (1:10) Normal mammary gland 1:10 Decloaking chamber Vimentin IgG1 (1:10) Normal mammary gland 1:200 Decloaking chamber Desmin IgG1 (1:10) Normal mammary gland 1:100 Decloaking chamber Smooth muscle actin (SMA) IgG2a (1:100) Normal mammary gland 1:100 Decloaking chamber Neurofilament (NF) IgG1 (1:25) Normal spinal cord 1:400 Decloaking chamber p63 IgG2a (1:100) Normal mammary gland 1:300 Decloaking chamber Myosin (M4276) IgG1 (1:17,5) Normal skeletal muscle 1:700 Decloaking chamber CD 31 Polyclonal goat (normal rabbit serum)
Normal mammary gland
1:500 Decloaking chamber
Type I collagen IgG1 (1:300)
Normal cartilage and bone (rib joint)
1:3000 Pepsin
Type II collagen IgG1 (1:500)
Normal cartilage and bone (rib joint)
1:5000 Pepsin
Osteopontin Polyclonal rabbit (normal goat serum)
Normal cartilage and bone (rib joint)
1:3000 None
The stability of intermediate filaments (cytokeratin, vimentin, desmin, NF) makes it possible to characterise and study the histogenesis of different tumours by the use of monoclonal antibodies against intermediate filaments. 6
4.4.1 Monoclonal mouse anti-human cytokeratin clones AE1/AE3
Clone AE1 and AE3 Code No. M 3515
Lot 072. Edition 10.22.01
Immunogen: human epidermal callus
The cytokeratins are a family of water-soluble proteins with molecular weights beween 40-70 kDa that form the cytoskeleton of epithelial cells. At least 19 different cytokeratins have been identified and can be divided into two subfamilies. Subfamily A comprises relatively acidic cytokeratins with a pI under 5.5, whereas members of subfamily B have a relatively basic pI of 6 or over.
The monoclonal mouse antibody is provided in a 1,0 ml size as purified immunoglobulin fraction of ascites in 0,05 mol/l Tris/HCl, pH 7,2 containing 15 mmol/l NaN3.
Isotype: IgG1 kappa
Mouse Ig concentration: 207 µg/ml Total protein concentration: 11,2 mg/ml
AE1/AE3 is a cocktail of two monoclonal antibodies that were obtained by immunizing mice with human callus keratins. Antibody AE1 immunoreacts with an antigenic determinant present on most of the subfamily A cytokeratins, including No. 10, 13, 14, 15, 16 and 19 but no No. 12, 17 and 18. Antibody AE3 reacts with an antigenic determinant shared by the subfamily B cytokeratins, including No. 1, 2, 3, 4, 5, 6, 7 and 8.
In normal tissues, the antibody stains the epithelial cells, the luminal and the myoepithelial (figure 7).
Figure 7: AE1/AE3 (epithelial cells, red) in normal dog mammary gland tissue
4.4.2 Monoclonal mouse anti-human vimentin
Clone V 9
Code No. M 0725
Lot 092. Edition 16.09.02.
Vimentin is a 57 kDa intermediate filament (IF) protein, which forms part of the cytoskeleton of vertebrate cells. The cell specifity, displayed by each of the IF subtypes, is retained in malignant cells as well as their normal counterpart, which makes IF´s important as diagnostic markers in histogenesis of tumour cells.
The monoclonal mouse antibody is provided in liquid form as cell culture supernatant dialysed against 0,05 mol/l Tris/HCl, pH 7,2 containing 15 mmol/l NaN3.
Isotype: IgG1 kappa
Mouse Ig concentration: 360 mg/l Total protein concentration: 19,3 g/l
In abnormal tissue, the antibody labels 17/20 sarcomas, 16/18 melanomas and 3/3 Schwannomas. In addition, variable percentages (10-57%) of carcinomas, neuroendocrine carcinomas, neuroblastomas, thymomas, mesotheliomas are positive with the antibody. With the exception of the neuroblastomas, cytokeratin is coexpressed with vimentin in these tumours. 35
Figure 8: Vimentin (red) in normal dog mammary gland tissue
4.4.3 Monoclonal mouse anti-human neurofilament
Clone 2F11 Code No. M 0762
Lot 071. Edition 04.03.03.
The antibody labels neurons (axons and dendrites) of the central and peripheral nervous system and is a useful tool for the identification of tumours with neuronal differentiation (figure 9). Neurofilaments (NF’s) belong to the family of intermediate filaments (IF) and are structural elements of the neuronal cytoskeleton. Cells labelled by the antibody, display a cytoplasmic staining pattern.
The monoclonal mouse antibody is provided in liquid form as cell culture supernatant dialysed against 0,05 mol/l Tris/HCl, pH 7,2 containing 15 mmol/l NaN3. 36
Isotype: IgG1 kappa
Figure 9: Neurofilament (neurons, red) in normal dog spinal cord
4.4.4 Monoclonal mouse anti-human type I collagen
Clone number: COL-1 Product code: ab6308 Isotype: IgG1
Figure 10: Collagen type I (osteoblasts, red) in normal dog rib joint
4.4.5 Monoclonal mouse anti-human type II collagen
Catalog number : MAB 1330 Concentration: 1 mg/ml Lot number: 24050155 Clone name: COLL-11 Isotype: Ms IgG1 kappa
Figure 11: Collagen type II (cartilage, red) in normal dog rib joint
4.4.6 Monoclonal mouse ascites fluid anti-skeletal myosin
Clone number: MY-32 Product number: M-4276 Lot number: 036H4835
The antibody is used for detecting cross striated muscle differentiation in tumours. The antibody localizes an epitope on the myosin chain that is stable to the routine formalin-fixation and paraffin-embedding process. The antibody is specific for the myosin heavy chain in skeletal muscle tissue (figure 12).
Myosin is a protein known to interact with actin in muscle and in non-muscle cells. It contains two identical heavy chains and four light chains. Myosin molecules consist of two major regions: tails and heads. They aggregate into filaments through the tail region and interact with actin and with ATP through the head region.
The product is provided as ascites fluid with 0,1 % sodium azide as a preservative. 39 Isotype: Mouse Ig G1
Figure 12: Myosin (striated muscle cells, red) in normal dog muscle tissue
4.4.7 Monoclonal mouse anti-human desmin
Clone: D33
Code number: M 0760 Lot number: 048
The antibody reacts with the 53 kDa intermediate filament protein Desmin in human muscle cells. The antibody shows a broad interspecies cross-reactivity reacting with Desmin from a variety of other species. The antibody does not recognize other filament proteins.
The monoclonal mouse antibody is supplied in liquid form as tissue culture supernatant, dialysed against 0,05 mol/l Tris/HCl, pH 7,2 and containing 15 mmol/l NaN3.
Mouse Ig concentration: 240 mg/l Isotype: IgG1, kappa
Total protein concentration: 16,1 g/l
Immunogen: desmin purified from human muscle
In normal tissue, both striated (skeletal and cardiac) and smooth muscle cells are labelled (figure 13). The labelling is confined to the Z bands in skeletal and cardiac muscle, giving a characteristic striated appearance.
Figure 13: Desmin (muscle cells of artery, red) in normal dog mammary gland tissue
4.4.8 Monoclonal mouse anti-human smooth muscle actin
Clone: 1 A4
Code number: M 0851 Lot number: 028
Smooth muscle actin is commonly used and has been established as a specific and sensitive marker. However, sometimes the staining can not be interpreted, since actin stains stromal fibroblasts and vascular smooth muscle cells as well.
The monoclonal mouse antibody is supplied in liquid form as tissue culture supernatant, dialysed against 0,05 mol/l Tris/HCl, pH 7,2 and containing 15 mmol/l NaN3.
Mouse Ig concentration: 75 mg/l Isotype: IgG2a, kappa
Total protein concentration: 3,1 g/l
Immunogen: N-terminal decapeptide of human α-smooth muscle actin
The antibody reacts with smooth muscle cells of vessels and different parenchymes without exception, but with different intensity, according to the amount of α-smooth muscle actin present in smooth muscle cells, myoepithelial cells, pericytes, and some stromal cells in the intestine, testis, breast and ovary.
Smooth muscle actin is commonly used to stain mammary myoepithelial cells, whose presence or absence is a reliable criterion for differentiating in situ and invasive carcinomas (figure 14). However, some morphologically distinct myoepithelial cells fail to stain. These smooth muscle actin- negative myoepithelial cells might be newly formed through stem cell-mediated proliferation and are in the transition to a terminally differentiated status. The myoepithelial cells might also be subject to different normal and pathological changes, and those might significantly affect the functions and biological behaviour of the adjacent epithelial cells. 41, 42, 43
Figure 14: Smooth muscle actin (cytoplasm myoepithelial cells, red) in normal dog mammary gland tissue
4.4.9 Monoclonal mouse anti-human p63 protein
Clone: 4A4
Code number: M 7247 Lot number: 00001797 Edition: 16.06.03
p63 may be a useful tool for the differential diagnosis of benign versus malignant prostatic lesions, for the identification of squamous cell carcinomas and as a specific marker of metaplastic carcinoma of the breast. It is highly specific since neither stromal fibroblasts nor vascular smooth muscle cells are stained.
The p63 protein is a member of the p53 family, which also includes p73. At least 6 different transcripts of p63 derive from alternative splicing events. The predominant localization of p63 protein is in the basal layer of stratified squamous and transitional epithelia. These basal cells act as the progenitors of the suprabasal cells, which undergo differentiation and cell death in regenerative epithelia.
Monoclonal mouse antibody is supplied in liquid form as cell culture supernatant, dialysed against 0,05 mol/l Tris/HCl, pH 7,2 and containing 15 mmol/l NaN3.
Mouse IgG concentration: 375 mg/l Isotype: IgG2a, kappa
Total protein concentration: 15,0 g/l
Cells labelled by the antibody, display a nuclear staining pattern.
In normal tissues, the antibody strongly labels proliferating basal cells of epithelial layers in the epidermis, foreskin, uterine cervix, vagina, urothelium, and prostate, whereas secretory cells and neuroendocrine cells in the prostate are negative. In the lung, the antibody labels basal and suprabasal cells in the bronchial mucosa, and myoepithelial cells of seromucous glands.
In normal parotid tissue the expression of p63 is restricted to few basal and myoepithelial cells. Ductal luminal and acinus cells are completely negative.
In abnormal tissue, the salivary gland tumours show a strong nuclear staining. No staining has been noted in infiltrative carcinomas. Also the stromal cells were unreactive to p63. 43, 44, 45, 46, 47
4.4.10 Mouse IgG2a negative control
Code No.: X 0943 Lot number: 129 Edition: 06.08.02.
The reagent is a mouse monoclonal IgG2a antibody (clone DAK-G05) supplied in liquid form as tissue culture supernatant, dialysed against 0,05 mol/l Tris/HCl, 15 mmol/l NaN3, pH 7,2.
Protein concentration: 9,5 g/l Mouse Ig concentration: 200 mg/l Isotype: IgG2a, kappa
The antibody specifity is directed towards Aspergillus niger glucose oxidase, an enzyme which is neither present nor inducible in mammalian tissues. 48
4.4.11 Mouse IgG1 negative control
Code number: X 0931 Edition: 01.05.03
It is especially used as a negative control for monoclonal mouse antibodies of isotype Ig G1.
The antibody is provided in liquid form as cell culture supernatant, dialysed against 0,05 mol/l Tris/HCl, pH 7,2 and containing 15 mmol/l NaN3.
Isotype: IgG1, kappa
Mouse IgG1 concentration: 100 mg/l Total protein concentration: 19,2 g/l
Figure 16: Negative control in normal dog mammary gland tissue
4.4.12 Polyclonal goat CD 31 PECAM-1 (M-20): sc-1506
CD 31 (or PECAM-1) is a glycoprotein expressed on the cell surfaces of monocytes, neutrophils, platelets and a subpopulation of T cells. In mammary gland tissue, CD31 stains the endothelial cells (figure 17).
Cell adhesion molecules are a family of closely related cell surface glycoproteins involved in cell-cell interactions during growth and are thought to play an important role in embryogenesis and development.
Figure 17: CD 31 (endothelial cells, red) in normal dog mammary gland tissue
4.4.13 Polyclonal Rabbit Anti-Osteopontin
Osteopontin is an arginine-glycine-aspartic acid containing a glycoprotein that interacts with integrins and CD 44 as major receptors. Osteopontin is multifunctional with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function and control of tumour cell phenotype. It has a unique functional role during tissue injury and stress.
Figure 18: Osteopontin (red) in normal dog rib joint
4.5 TUMOURS
In this project, two primary mammary tumours were studied. Cell lines were established from these tumours and they were grown in nude mice (table 2).
Table 2: The primary dog tumours and the mouse tumours used in the project
Primary tumours (from the dog) Mouse tumours
5 RESULTS AND DISCUSSION
5.1 RESULTS AND DISCUSSION OF THE PRIMARY DOG TUMOURS
The results of the primary dog tumours are ordered in table 3. The antibodies are compared with each other on different tumour tissues. The following legend is helpful to interpretate the results:
+++: almost all the cells are stained (> 50 %) ++: cells are stained but not all (20-50 %) +: only a few single cells are stained -: the cells are negative
/: the tissue section was not studied by the antibody
Table 3: Evaluation of two primary tumours that were stained with 8 antibodies
PRIMARY TUMOURS SMA (1:100) p63 (1:300) AE1/AE3 (1:10) Vimentin (1:200) Desmin (1:200) Myosin (1:700) NF (1:400) CD 31 (1:500) 353 B P 91/94 - - + +++ - - + / 353 H4 P 91/94 Cells: - Stroma: + Cells: + Stroma: - Cells: +++ Stroma: - Cells: ++ Stroma:+ - - - +++ Discussion
The cells of the osteosarcoma 353 B P 91/94 were very strongly positive for vimentin. Only a few cells were stained with AE1/AE3 and NF. And the tumour was negative for desmin, myosin, SMA and p63.
Figure 19: Positivity for CD 31 (endothelial cells, red) in tumour 353 H4 P91/94
5.2 RESULTS AND DISCUSSION OF THE MOUSE CARCINOMAS
The staining results of the mouse carcinomas (353 H4) are shown in table 4.
+++: almost all the cells are stained (> 50 %) ++: cells are stained but not all (20-50 %) +: only a few single cells are stained -: the cells are negative
