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Biotechnic & Histochemistry

ISSN: 1052-0295 (Print) 1473-7760 (Online) Journal homepage: http://www.tandfonline.com/loi/ibih20

Optimization, validation, and identification of

two reliable antibodies for immunodetection of

WNT5A

Z Prgomet, T Andersson & P Lindberg

To cite this article: Z Prgomet, T Andersson & P Lindberg (2017) Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A, Biotechnic & Histochemistry, 92:1, 46-58, DOI: 10.1080/10520295.2016.1255995

To link to this article: http://dx.doi.org/10.1080/10520295.2016.1255995

Published with license by Taylor & Francis Group, LLC© 2017 Z Prgomet, T Andersson, P Lindberg

Published online: 03 Feb 2017.

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Optimization, validation, and identi

fication of two

reliable antibodies for immunodetection of WNT5A

Z Prgomet

1,2

, T Andersson

2

, P Lindberg

1 1

Oral Pathology, Faculty of Odontology, Malmo University, Malmo, Sweden and2Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Clinical Research Centre, Skane University Hospital, Malmo, Sweden

Accepted October 29, 2016

Abstract

WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analy-sis. Validation of the primary antibodies, however, is often neglected. We characterized anti-bodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.

Key words: antibodies, immunohistochemistry, optimization, pre-absorption test, validation, wsestern blot analysis, WNT5A

WNT5A is a member of the WNT family; it is a cysteine-rich secreted signaling protein that is essen-tial for embryonic development and tissue homeosta-sis. WNT5A participates in regulating proliferation, differentiation, polarity, survival, and migration; it also plays a critical role in cancer progression (Camilli and Weeraratna 2010, Kikuchi et al. 2012, Nishita et al. 2010). Like other WNT proteins,

WNT5A is post-translationally glycosylated and pal-mitoylated. These modifications are essential for its secretion and signaling properties (Kurayoshi et al.

2007). Although WNT5A activates primarily the non-canonical WNT/planar cell polarity and WNT/Ca2+ signaling pathways, it also can inhibit or activate the canonical WNT/β-catenin pathway. The nature of WNT5A activity likely depends on the receptor and type of cell with which it interacts (Kim et al.2015, Li et al. 2010, Ring et al. 2014, Roarty et al. 2009). For example, WNT5A acts as a cancer suppressor in breast (Prasad et al.2013), colon (Dejmek et al.2005), and thyroid cancer (Kremenevskaja et al.2005), but as a cancer promoter in malignant melanoma (Jenei et al.

2009), oral squamous cell carcinoma (Prgomet et al. 2014), and pancreatic cancer (Bo et al.2013). For accu-rate prediction of cancer progression and prognosis, clinical pathologists require tissue biomarkers that accurately reflect the status of the disease (O’Hurley et al.2014, Schuster et al.2012); WNT5A potentially is one such biomarker.

Correspondence: Pia Lindberg, Oral Pathology, Faculty of Odontology, Malmö University, SE-20506 Malmö, Sweden. Phone: +46406657504, fax: +46406658490, e-mail:pia.lindberg@mah.se

Color versions of one or more of thefigures in the article can be found online atwww.tandfonline.com/ibih

Published with license by Taylor & Francis Group, LLC © 2017 Z Prgomet, T Andersson, P Lindberg

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Immunohistochemistry (IHC) commonly is used for clinical pathology, particularly for cancer diagnostics when the tumor is poorly differen-tiated (Gremel et al. 2014, Hewitt et al. 2014). Immunohistochemical identification of WNT5A in cancer tissues would be a valuable diagnostic tool. Currently, however, IHC lacks accuracy, reprodu-cibility and reliability. Limitations of IHC may be due, in part, to insufficient validation of the speci-ficity of the primary antibody (Holmseth et al.

2012, O’Hurley et al.2014, Schuster et al.2012). Various commercially available antibodies have been used for detecting WNT5A by IHC and wes-tern blot analysis in both cell lines and tissue sam-ples. The specificity of the WNT5A antibodies used is uncertain, however, and rarely reported (Syed Khaja et al. 2011). Therefore, we validated and characterized commercially available WNT5A anti-bodies that would be suitable for detecting WNT5A by IHC or western blot analysis.

Material and methods

Cell lines, chemicals, and peptides

Our quality assurance study was approved by the Faculty of Odontology, Malmo University, Malmo, Sweden. The human mammary carcinoma cell line, MDA-MB468 (Lot no. 58683213), was purchased from American Type Culture Collection (Manassas, VA). It was transfected with either an empty vector (468-EV) or a WNT5A-containing vector (468-5A) and main-tained as described previously (Prasad et al. 2013). Recombinant WNT5A (rWNT5A; 645-WN) and recombinant WNT3A (rWNT3A; 1324-WN) were purchased from R&D Systems (Minneapolis, MN). Antibody diluent (S2022); peroxidase blocking reagent (HPBK; S2023); the secondary antibodies

rabbit anti-goat/HRP (P0449) and goat anti-mouse/ HRP (P0447) and an ENVISION kit (goat anti-mouse/ rabbit/HRP; K5007) were purchased from DAKO (Glostrup, Denmark). Normal horse serum (2.5%; S-2012); 3,3´-diaminobenzidine (DAB; SK-4105); and secondary antibodies horse anti-goat/HRP (ImmPRESS reagent; MP-7405) and unconjugated rabbit anti-goat antibody (AI-5000) were purchased from Vector Laboratories (Burlingame, CA). Mouse monoclonal α-tubulin antibody (DM1A; SC-32293) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Background sniper (BS966L10) was purchased from Biocare Medical (Concord, CA), and bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO).

WNT5A antibodies

Polyclonal goat anti-mouse/rat WNT5A (AF645) was purchased from R&D Systems, monoclonal mouse anti-human WNT5A clone 3A4 (H00007474-M04) was purchased from Abnova Corp. (Taipei, Taiwan), monoclonal mouse anti-human WNT5A clone 3D10 (NBP1-47438) was purchased from Novus Biologicals (Littleton, CO), and monoclonal mouse anti-human WNT5A clone 6F2 (LS-B3859) was purchased from LifeSpan BioSciences (Seattle, WA). These antibodies are here referred to as AF645, 3A4, 3D10, and 6F2, respectively. Antibodies were stored and handled according to the manufacturer’s instructions. We used different batches of the WNT5A antibodies for our study. Characteristics of the antibodies that were studied are given inTable 1.

Tissue samples

Archived samples of normal breast, breast cancer, oral squamous cell carcinoma (OSCC), and placenta

Table 1. Characteristics of WNT5A antibodies

Antibody Source Immunogen Purification

AF645

(R & D systems)

Polyclonal

goat anti-mouse/rat

E. coli-derived recombinant mouse WNT5A-peptide 254–334 aa Antigen affinity-purified 3A4 (Abnova) Monoclonal mouse anti-human

Partial recombinant WNT5A protein with Glutathione S-transferase tag 201–300 aa

Purified 3D10 (Novus Biologicals) Monoclonal mouse anti-human

Purified E. coli-derived recombinant fragment of WNT5A

Ascites

6F2 (LifeSpan Biosciences)

Monoclonal mouse anti-human

Purified E. coli-derived recombinant fragment of WNT5A

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tissues served as positive controls for validation of WNT5A antibodies because these tissues have been shown to express WNT5A (Diaz Prado et al.2009, Fracalossi et al.2010, Saitoh et al.2002, Sand-Dejmek et al.2013, Takahashi et al.2014, Zhong et al.2016). Normal liver tissue served as the negative control for WNT5A expression because it does not express WNT5A (Li et al.2014, Liu et al.2008).

Optimization of IHC

WNT5A antibodies were validated using 3 µm thick sections of formalin-fixed, paraffin-embedded tissues. Sections were deparaffinized and rehydrated, then subjected to heat-induced antigen retrieval (HIAR) at 95° C in a Decloaking ChamberTM (NxGen, Biocare Medical, Concord, CA). Two buffers, 10 mM citrate buffer, pH 6.0 and TRIS-EGTA buffer (10 mM TRIS, 0.5 mM EGTA), pH 9.0, were evaluated for HIAR. After cooling and washing with TRIS-buffered saline containing 0.1% Tween 20, pH 7.6 (TBS-T), nonspecific background staining was blocked with background sniper, 3% BSA in TBS-T or 2.5% normal horse serum. To obtain minimal background staining, background sniper was added for 10 min, and 2.5% normal horse serum for 30 min at room temperature, according to the manufacturer’s instructions, while 3% BSA in TBS-T was added for 30 min at room temperature, which was optimized by our laboratory. The sections then were incubated for 20 min at room temperature or overnight at 4° C with primary antibodies diluted with 2.5% normal horse serum, 1.5% BSA in TBS-T, or antibody diluent. The optimal dilution of antibo-dies was obtained by serial dilution experiments. Endogenous peroxidase was blocked for 10 min with HPBK before adding secondary antibodies: ENVISION, horse anti-goat, or rabbit anti-goat for 20 or 30 min at room temperature. We optimized time points in our laboratory to obtain distinct staining with minimum background. The immunoreaction was then visualized using DAB. The tissue sections were counterstained with hematoxylin, dehydrated, and mounted. After each step, the slides were washed three times for 3 min each with TBS-T. Omission of the primary antibody was used as an additional negative control for the secondary antibody. The images were obtained using a Nikon Eclipse 80i light microscope (Nikon Corp., Tokyo, Japan) at 400 ×.

Western blot analysis

MDA-MB468 and 468-EV cell lysates served as nega-tive controls, and MDA-MB468 cell lysate supple-mented with 4 ng/µl of rWNT5A and 468-5A cell lysate served as positive controls (Prasad et al.2013,

Prgomet et al.2015). Cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 30 mM sodium pyrophosphate, 1 mM EDTA, 1.5 mM MgCl2, 0.1 mM sodium orthovandate, 10%

gly-cerol, 1% Triton X-100) supplemented with protease inhibitors (one tablet of complete mini EDTA-free and one tablet of PhosSTOP, both from Roche Diagnostics, Mannheim, Germany) for 30 min on ice and centrifuged at 18,000 × g for 30 min at 4° C. The supernatant was then used as the cell lysate. The protein content of each cell lysate was assessed using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA).

For reducing western blot analysis, the cell lysates were prepared in 4 × NuPAGE LDS Sample Buffer (NP0007; Life Technologies, Carlsbad, CA) supplemented with 200 mM dithiothreitol (DTT) and boiled. Thirty µg of total protein was loaded onto 10% sodium dodecyl sulfate polyacrylamide electrophoresis gel (SDS-PAGE) followed by semi-dry blotting onto a PVDF membrane for 1 h. For native western blot analysis, the cell lysates were prepared in 2 × Tris-glycine native sample buffer (200 mM Tris, 20% glycerol, 0.005% bromophenol blue), pH 8.6, and 30μg total protein was loaded on 10% PAGE without SDS. After electrophoresis, the native blots were incubated in 0.1% SDS for 15 min before semi-dry blotting onto a PVDF membrane for 1 h, excluding SDS. The blots then were blocked in 3% (w/v) non-fat skimmed milk powder (06-019; Scharlab S.L., Barcelona, Spain) in TBS-T for 1 h and incubated with primary antibodies diluted 1:100 in 3% (w/v) non-fat skimmed milk powder in TBS-T overnight at 4° C. After washing with TBS-T, the blots were incubated with either rabbit anti-goat/ HRP or goat anti-mouse/HRP diluted in 5% (w/v) non-fat skimmed milk powder in TBS-T for 1 h at room temperature. The immunoreaction was devel-oped with a chemiluminescence HRP substrate. α-Tubulin antibody was used as the loading control.

Purification of 3D10 antibody

The 3D10 monoclonal antibody was purified using PierceTM Protein A/G magnetic beads (88802; Thermo Scientific) according to the manufacturer’s instructions. The purified antibody was assessed by both IHC and western blot analysis using the same protocols as the unpurified 3D10 antibody. Pre-absorption test of primary antibody specificity for western blot analysis and IHC A pre-absorption test was performed prior to IHC and western blot analysis. The diluted antibodies

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were incubated overnight at 4° C with 10 × molar excess of rWNT5A or rWNT3A before application to the tissues and blots. For western blot, the pre-absorption test was performed on two blots loaded with aliquots from the same samples. One blot was probed with the antibody–antigen complex and the other with the primary antibody alone. MDA-MB468 cell lysate supplemented with 4 ng/µl of rWNT3A was used as a specificity test for the polyclonal AF645 antibody for western blot analysis.

Evaluation of the pre-absorption test for IHC was performed in two steps. First, it was ascer-tained that identical areas were immunosascer-tained in two consecutive sections used for the test. The results revealed that the stained area of each pair of tissue sections, calculated as IHC stained area (DAB)/total area, was identical. Staining intensi-ties then were compared for each pair of tissue sections stained with primary antibody alone or with primary antibody that had been pre-incu-bated with rWNT5A or rWNT3A. The stained areas and the staining intensities were evaluated using ImageJ software (NIH, Bethesda, MD).

Results

Optimization of IHC protocol

IHC protocols were evaluated with regard to HIAR method, nonspecific background blocking solution, primary antibody dilution, incubation time, and the secondary antibody. Better morphology was

observed when HIAR was performed with 10 mM citrate buffer, pH 6.0, than with the TRIS-EGTA buffer, pH 9.0, and the optimal HIAR pro-tocol was 95° C for 40 min (data not shown). Results varied when different combinations of non-specific background blocking solutions and sec-ondary antibodies were used with the primary antibodies. For example, high nonspecific back-ground staining with the polyclonal AF645 anti-body was observed when BSA blocking solution was used in combination with the unconjugated linker secondary antibody (rabbit anti-goat) and ENVISION (goat anti-mouse/rabbit/HRP) (data not shown). This problem was circumvented using a polymer horse goat secondary anti-body for detecting the polyclonal AF645 antianti-body, while ENVISION was used for detecting the mono-clonal antibodies. Table 2 shows the optimized IHC protocols.

Immunostaining patterns for WNT5A protein expression

Different immunostaining patterns for WNT5A expression were observed in the tissues we stud-ied, and the patterns varied with the antibody used. The tissues were from placenta, normal breast, breast cancer, OSCC and normal liver. When the polyclonal AF645 antibody was used, mostly perinuclear and cytoplasmic, but also some nuclear WNT5A immunostaining was observed in trophoblast cells of placenta, ductal

Table 2. Optimized IHC protocols for WNT5A antibodies: polyclonal AF645 antibody (R & D), the monoclonal 3A4 (Abnova), 3D10 (Novus Biologicals), and 6F2 (LSBio)

Antibody AF645 3A4 3D10 6F2

Antigen retrieval Citrate buffer, pH 6.0, 95° C, 40 min Citrate buffer, pH 6.0, 95° C, 40 min Citrate buffer, pH 6.0, 95° C, 40 min Citrate buffer, pH 6.0, 95° C, 40 min Background block 2.5% horse serum, 30 min Background sniper, 10 min Background sniper, 10 min Background sniper, 10 min Antibody dilution 1:100 in 2.5% horse serum overnight 4° C 1:75 in antibody diluent (DAKO) overnight 4° C 1:20,000 in antibody diluent (DAKO) 20 min room temperature 1:30,000 in antibody diluent (DAKO) 20 min room temperature Peroxidase block Peroxidase blocking reagent (HPBK), 10 min Peroxidase blocking reagent (HPBK), 10 min Peroxidase blocking reagent (HPBK), 10 min Peroxidase blocking reagent (HPBK), 10 min Secondary antibody

ImmPRESS horse anti-goat/HRP, 30 min ENVISION, 20 min ENVISION, 20 min ENVISION, 20 min

Substrate DAB DAB DAB DAB

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epithelial cells of normal breast, breast cancer, and OSCC tissues (Fig. 1A–D). When the three mono-clonal antibodies, 3A4 (Fig. 1F–I), 3D10 (Fig. 1K– N), and 6F2 (Fig. 1P–S), were used, however, only cytoplasmic WNT5A staining was apparent in the same tissues. We found no immunostaining for WNT5A with the 3A4 antibody in liver tissue (Fig. 1J), but there was distinct cytoplasmic stain-ing with the other two monoclonal antibodies, 3D10 (Fig. 1O) and 6F2 (Fig. 1T). When the

polyclonal AF645 antibody was used, we observed weak cytoplasmic, perinuclear, and some nuclear staining for WNT5A in the hepatocytes (Fig. 1E).

Validation of IHC by western blot analysis Based on different staining patterns, the antibodies were assessed by western blot analysis using cell lysates that were confirmed to be either WNT5A-positive or WNT5A-negative. The polyclonal

Fig. 1. IHC expression of WNT5A. A–E) Perinuclear, cytoplasmic, and in some cells nuclear immunostaining with the polyclonal AF645 antibody. F–T) Cytoplasmic staining of WNT5A with the monoclonal antibodies (F–I) 3A4, (K–N) 3D10, and (P–S) 6F2. J) Lack of the WNT5A staining in hepatocytes of normal liver with the monoclonal 3A4 antibody. O) Cytoplasmic staining of WNT5A with the monoclonal 3D10 and 6F2 (T) antibodies. Scale bars = 50 µm.

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AF645 antibody identified a 43-kDa band that cor-responded to the WNT5A protein in the WNT5A-positive cell lysates, but not in the WNT5A-nega-tive cell lysates (Fig. 2A). The other three antibo-dies detected several bands that corresponded to lower or higher molecular weights than 43 kDa, which is the molecular weight of WNT5A, in dif-ferent cell lysates (Fig. 2B–D). Native western blot

analysis was performed to determine whether the monoclonal antibodies could detect WNT5A under nonreducing conditions. No bands were detected with the polyclonal AF645 or the monoclonal 3A4 antibodies (data not shown). When the other two monoclonal antibodies, 3D10 and 6F2, were used, multiple bands were present, but none was close to 43 kDa (Fig. 3A, B).

Fig. 2. Assessment of IHC using reducing western blot analysis. A) WNT5A was detected at 43 kDa in MDA-MB468 cell lysate supplemented with 4 ng/µl of rWNT5A and MDA-MB468 cells transfected with a WNT5A-containing vector, with the polyclonal AF645 antibody. B–D) No WNT5A protein band was detected with the monoclonal antibodies (B) 3A4, (C) 3D10 or (D) 6F2.α-Tubulin antibody was used as the loading control.

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Purification of 3D10 antibody

Two of the monoclonal antibodies used here, 3D10 and 6F2, were unpurified antibody-rich fluids (ascites). The 3D10 antibody was purified as described for the validation of the immunoreactions obtained by IHC and western blot analysis. The pur-ified 3D10 antibody showed an identical cytoplasmic immunostaining pattern for normal breast, breast cancer, and OSCC tissue sections (Fig. 4A) as the unpurified 3D10 antibody in the same tissue sections shown in Fig. 1L–N. Neither the purified nor the

unpurified 3D10 antibody detected WNT5A in cell lysates, however (Figs. 2C,4B).

Specificity of immunoreactivity

A pre-absorption test was used to examine the specificity of three antibodies: AF645, 3A4, and purified 3D10. The intensity of the cytoplasmic, perinuclear, and nuclear immunostaining of WNT5A-positive tissue sections by polyclonal AF645 antibody was not reduced by pre-incuba-tion with rWNT5A (Fig. 5A, B) or with rWNT3A (Fig. 5C,D).

When the monoclonal 3A4 antibody was pre-incubated with rWNT5A, the intensity of the cyto-plasmic immunostaining was reduced by 89% (Fig. 6A,B) and by 39% when the same antibody was pre-incubated with rWNT3A (Fig. 6C, D). These results are not surprising because WNT5A and WNT3A possess 45% homology overall in

their amino acid sequence. Considering only the amino acid sequence against which the 3A4 anti-body is directed (amino acids 201–300), the simi-larity between the WNT3A and WNT5A is 50% according to a Basic Local Alignment Search Tool (BLAST) analysis (Blast 2015) (data not shown). The analysis of the purified monoclonal 3D10 anti-body that had been pre-incubated with rWNT5A exhibited a 32% increase in the intensity of the cytoplasmic immunostaining of the WNT5A-posi-tive tissue sections (Fig. 6E,F).

Immunoblotting of WNT5A-positive cell lysates was clearly reduced when the polyclonal AF645 antibody had been pre-incubated with rWNT5A (Fig. 7A, B). Moreover, the polyclonal AF645 antibody did not detect WNT3A in the MDA-MB468 cell lysate that was supplemented with rWNT3A; however, it detected WNT5A in WNT5A-positive cell lysates as reported pre-viously (Fig. 7C).

Discussion

IHC commonly is used for clinical diagnosis and biomedical research, but there are no standardized guidelines for validation of primary antibodies (Hewitt et al. 2014, O’Hurley et al. 2014, Voskuil

2014). The antibodies that we investigated for IHC and western blot analysis have been used in pub-lished reports and were selected randomly from different companies. We evaluated the antibodies

Fig. 3. Assessment of IHC with native western blot analysis. A, B) Lack of the WNT5A protein band at 43 kDa in the MDA-MB468 cell lysate supplemented with 4 ng/µl of rWNT5A and the MDA-MB468 cell lysate transfected with a WNT5A-containing vector with two monoclonal antibodies (A) 3D10 and (B) 6F2.

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according to published recommendations for this purpose (Hewitt et al.2014, O’Hurley et al.2014).

The outcome of IHC is influenced by many fac-tors including the HIAR method, the nonspecific background blocking solution, dilution of the pri-mary antibody and the choice of secondary antibody. We optimized all these parameters for each antibody investigated. We observed much nonspecific back-ground staining when BSA was used as the blocking reagent before adding the primary polyclonal goat AF645 antibody, the unconjugated linker secondary rabbit anti-goat, and ENVISION. There are several possible cross-reactions that could account for the high background staining: between the two second-ary antibodies; between the primsecond-ary antibody and

ENVISION, because they both are produced in goats; or between BSA and the primary antibody. Therefore, the polyclonal AF645 antibody should not be used with BSA, the unconjugated linker sec-ondary antibody (rabbit anti-goat), or ENVISION (goat anti-mouse/rabbit/HRP); it should be used only with a conjugated anti-goat secondary antibody.

Appropriate positive and negative controls are crucial for antibody validation. We used tissues that have been demonstrated by quantitative polymerase chain reaction (qPCR), western blot, or IHC either to express or lack expression of WNT5A as positive and negative controls. Omission of the primary antibody was used as additional control to ensure that the

Fig. 4. Detection of WNT5A with the purified 3D10 antibody. A) Cytoplasmic immunostaining of WNT5A protein with the purified 3D10 antibody in duct epithelial cells of normal breast tissue, breast cancer cells, and OSCC cells. B) Lack of WNT5A protein band at 43 kDa in the MB468 cell lysate supplemented with 4 ng/µl of rWNT5A and the MDA-MB468 cell lysate transfected with a WNT5A-containing vector with the purified monoclonal 3D10 antibody. α-Tubulin antibody was used as the loading control. Scale bars = 50 µm.

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immunostaining observed was not caused by tissue processing, blocking reagents, or cross-reactivity of the secondary antibodies. Normal liver tissue was used as the negative control for IHC staining, because it has been shown to possess undetectable mRNA and protein expression of WNT5A (Li et al.

2014, Liu et al.2008). The monoclonal 3A4 antibody was the only antibody that did not detect any cyto-plasmic WNT5A immunostaining in our negative control, but at the same time stained cytoplasmic WNT5A in our positive controls. Thesefindings are consistent with those of previously published studies of WNT5A expression (Diaz Prado et al. 2009, Fracalossi et al. 2010, Li et al.2014, Liu et al.2008, Saitoh et al.2002, Sand-Dejmek et al.2013, Takahashi et al.2014, Zhong et al.2016). To the contrary, the other three antibodies showed a variety of WNT5A immunostaining in all tissues. The monoclonal 3D10 antibody showed a strong cytoplasmic WNT5A immunostaining in all tissues including the normal liver tissue even after purification.

Immunostaining with the monoclonal 6F2 anti-body was consistent with considerable expression

of WNT5A in the cytoplasm of trophoblasts of placenta and in breast cancer tissue, while cyto-plasmic immunostaining for WNT5A of the hepa-tocytes of normal liver, ductal epithelial cells of normal breast tissue and OSCC tissue was weak. Immunostaining patterns using the polyclonal AF645 antibody indicated expression of WNT5A in the cytoplasm of placental trophoblasts, ductal epithelial cells of normal breast, breast cancer, and OSCC tissue, whereas the staining of the normal liver tissue was weak and present in only limited areas. The polyclonal AF645 antibody exhibited what appeared to be perinuclear staining and also nuclear staining in some cells. Other investi-gators (Da Forno et al. 2008, Fracalossi et al.2010) have reported nuclear expression of WNT5A with the same AF645 antibody and Da Forno et al. (2008) suggested that this staining pattern was nonspecific because the nuclear expression of WNT5A was unrelated to progression of mela-noma. Although we did not evaluate whether the perinuclear immunostaining pattern was related to the nuclei or it was an artifact as others

Fig. 5. Pre-absorption test of the polyclonal AF645 antibody for IHC. Intensity of the cytoplasmic, perinuclear and nuclear WNT5A immunostaining in normal breast tissue with the polyclonal AF645 antibody (A and C) was not decreased by pre-incubation with (b) rWNT5A or (d) rWNT3A. Scale bars = 50 µm.

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have reported, we did not observe this immunos-taining pattern with the monoclonal 3A4 anti-body. If this staining pattern is an artifact that is obtained only with the polyclonal AF645 anti-body, it could be due to effects of HIAR on the antigen–antibody binding activity, which is con-sistent with an earlier interpretation (Wieczorek et al. 1997). Regardless of the reason for the

perinuclear immunostaining pattern of the AF645 antibody, the phenomenon complicates interpreta-tion of IHC staining for WNT5A.

Because the antibodies we selected, except for monoclonal 3A4, were recommended by their manufacturers for IHC and western blot analysis, we investigated the expression of WNT5A in cell lysates of the human mammary carcinoma cell line,

Fig. 6. Pre-absorption test of the monoclonal antibodies in IHC. A, B) Intensity of the cytoplasmic WNT5A immunostaining in normal breast tissue with the monoclonal 3A4 antibody (A) was markedly reduced after pre-incubation with rWNT5A (B). C , D) The intensity of the cytoplasmic WNT5A staining in normal breast tissue with the monoclonal 3A4 antibody (C) was moderately decreased after pre-incubation with rWNT3A (D). E, F) The intensity of the cytoplasmic WNT5A staining in normal breast tissue with the purified monoclonal 3D10 antibody (E) was increased after pre-incubation with rWNT5A. Scale bars = 50 µm.

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MDA-MB468, using the different antibodies. We evaluated the monoclonal 3A4 antibody in parallel with other antibodies, although it is not recom-mended for application in western blot analysis. The band corresponding to 43 kDa, which is the size of the WNT5A protein, was visualized only by the polyclonal AF645 antibody. The nonspecific staining of this antibody in the IHC experiments is dealt with easily with western blot analysis because in western blot analysis, one can also

evaluate the size of the protein, e.g., 43 kDa for WNT5A. Also, no band corresponding to WNT5A was detected by the AF645 antibody in WNT5A-negative cell lysates, while it was present in WNT5A-positive cell lysates. It was not surprising that there was no 43-kDa band in western blot analysis with the monoclonal 3A4 antibody because it is not recommended for use in western blot analysis by its manufacturer. On the other hand, the 3D10 and 6F2 monoclonal antibodies

Fig. 7. Pre-absorption test of the polyclonal AF645 antibody in western blot analysis. A and B) Intensity of WNT5A band in the WNT5A-positive cell lysates (A) was reduced after pre-incubation of the AF645 antibody with rWNT5A (B). C) Lack of WNT3A protein band in the MDA-MB468 cell lysate supplemented with 4 ng/µl of rWNT3A, but strong detection of WNT5A protein at 43 kDa in WNT5A-positive call lysates with the AF645 antibody.α-Tubulin antibody was used as the loading control.

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that are recommended for detecting WNT5A in cell lysates could not distinguish between the WNT5A-negative and WNT5A-positive cell lysates; there was no protein band at the expected molecular weight of WNT5A, but there were protein bands at regions that corresponded to both lower and higher molecular weights. Purification of the 3D10 antibody did not alter the lower and higher protein bands that appeared in the western blot analysis. A likely explanation is that we used pro-tein A/G magnetic beads, which isolate all IgGs and not just IgGs for WNT5A protein. This means that the purified 3D10 antibody may contain other IgGs and therefore may recognize other proteins. The discrepancies we observed in detecting WNT5A by IHC and western blot analysis could also be explained by the appearance of different immunogenic epitopes. Formalinfixation of tissues would mask the immunogenic epitopes, while SDS-PAGE would denature the protein, which explains why western blot analysis is not always sufficient for validation of an antibody that is intended for IHC (Hewitt et al. 2014, O’Hurley

et al.2014, Schuster et al.2012).

The specificity of the immunoreactivity was assessed further using pre-absorption tests. Different results were obtained from the pre-absorption test of the polyclonal AF645 antibody with rWNT5A for IHC compared to western blot analysis. The reson for this is not clear, but a possible explanation is that antibody-rWNT5A complex binds to the tissue due to free antigen-binding sites of the antibody that binds to free epitopes in the tissue. Regardless of the reason for this binding, the discrepancy is another argu-ment that this antibody is less suitable for IHC. By contrast, the pre-absorption test with the monoclonal 3A4 antibody exhibited reduced intensity of cytoplasmic immunostaining in posi-tive control tissues, which makes it evident that this antibody binds specifically to WNT5A in for-malin-fixed tissues. Moreover, the AF645 and the 3A4 antibodies were tested for their specific detection of WNT5A using rWNT3A. The poly-clonal AF645 antibody did not detect rWNT3A in western blot analysis, which means that this anti-body is specific for detecting the WNT5A protein in western blot analysis. On the other hand, IHC detection of cytoplasmic WNT5A by the mono-clonal 3A4 antibody pre-incubated with rWNT3A was moderately inhibited (39%). This means that the monoclonal 3A4 antibody shows some cross-reactivity with the WNT3A protein, which was expected because of the similarity of the two WNT proteins. Also, the homology in the amino

acid sequence between the 3A4 antibody (201–300 aa) and rWNT3A (201–300 aa) is greater and shows 50% similarity. These findings, together with the more pronounced reduction of the inten-sity of the cytoplasmic immunostaining with the 3A4 antibody pre-incubated with rWNT5A (89%), suggest that the monoclonal 3A4 antibody is the best choice among the options we tested for detecting WNT5A by IHC.

Regardless of the guidelines from the manufac-turers, we found that different antibodies are suitable for different applications; therefore, similar studies should be performed using other commercially available WNT5A antibodies. Among the antibodies that we investigated, the monoclonal 3A4 antibody from Abnova is the most suitable for detection of WNT5A by IHC, while the polyclonal AF645 antibody from R&D is the best for western blot analysis of the WNT5A protein.

Acknowledgments

This work was supported by the Swedish Cancer Foundation, the Swedish Research Council, Skåne University Hospital Research Foundations, Gunnar Nilsson’s Cancer Foundation, the BioCare pro-gram, all to TA, and Malmö Allmänna Sjukhus Cancer Foundation to ZP and TA.

Declaration of interest: TA is a shareholder in WntResearch and part-time Chief Scientific Officer of WntResearch. This does not alter the author’s adherence to all policies on sharing data and materials as stated for Biotechnic & Histochemistry. ZP and PL report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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Figure

Table 1. Characteristics of WNT5A antibodies
Table 2. Optimized IHC protocols for WNT5A antibodies: polyclonal AF645 antibody (R & D), the monoclonal 3A4 (Abnova), 3D10 (Novus Biologicals), and 6F2 (LSBio)
Fig. 2. Assessment of IHC using reducing western blot analysis. A) WNT5A was detected at 43 kDa in MDA-MB468 cell lysate supplemented with 4 ng/µl of rWNT5A and MDA-MB468 cells transfected with a WNT5A-containing vector, with the polyclonal AF645 antibody

References

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