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Research Note

Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry

[version 1; peer review: 2 approved with reservations]
PUBLISHED 15 Oct 2014
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This article is included in the Antibody Validations gateway.

Abstract

ARID1A is a known suppressor of tumour formation and the Human Protein Atlas antibody HPA005456 has been demonstrated in previous literature to stain tumour tissue by immunohistochemistry (IHC) in formalin-fixed paraffin embedded human tissue and human cell lines. This article details the validation of this antibody for immunohistochemistry of formalin-fixed paraffin embedded murine tissue using a Leica BondMax immunostainer. Using Western blot and IHC on murine wild-type and knockout tissue we have demonstrated that this antibody to ARID1A correctly stains murine tissue by immunohistochemistry.

Keywords

immunohistochemistry, antibody, ARID1A, tissue

Introduction

ARID1A (AT-rich interactive domain 1a) is a member of the SWI/SNF family and its loss has been implicated as a factor in multiple premalignant and malignant conditions, including Barrett’s oesophagus and oesophageal carcinoma as well as endometrial and clear cell ovarian carcinomas and their precursor endometriotic lesions14. The ARID1A antibody from Human Protein Atlas is a rabbit antibody generated against a PrEST (Protein Epitope Signature Tag) fragment of the ARID1A gene and affinity purified against the same fragment5. It is thus designated as being “mono-specific” in that the affinity purification removes any non-specific or low affinity binders to the peptide. Through the Human Protein Atlas, the antibody has been tested on a wide variety of human tissue types and human malignancies, as well as for expression in immunofluorescence on U-2 OS, A-431 and U-251 MG cell lines. This demonstrates a nuclear expression in all cell lines and in the majority of tissue types6. However, the Western blot data were not supportive and did not produce staining corresponding to the expected size, although the data from a protein array did confirm a peak at the expected size. The antibody has been used to stain human colorectal cancers7, clear cell carcinomas8 and on a variety of clear cell cancer cell lines9 by immunohistochemistry.

To our knowledge, whilst the sequence homology between mouse and human ARID1A is 95%, this antibody has not been qualified using knockout tissue and has not been tested or published on murine tissue and this work represents the first data to do so.

Materials and methods

Reagent details

Details of all reagents with reference to the immunohistochemical staining procedure can be found in Table 1.

Table 1. Details of ancillary reagents for immunohistochemistry.

ProcessReagentManufacturerCatalogue NumberConcentration
FixationNeutral Buffered FormaldehydeSigmaHT50112810%
PretreatmentER1 (Sodium Citrate, pH6)Leica BiosystemsAR9961Proprietary
ER2 (Tris/EDTA, pH9)Leica BiosystemsAR9640Proprietary
Enzyme 1 (Proteinase K)Leica BiosystemsAR9551100 µg/ml
StainingPeroxide BlockLeica BiosystemsDS9263Proprietary
Streptavidin – HRPLeica BiosystemsDS9263Proprietary
Diaminobenizidine (DAB)Leica BiosystemsDS9263Proprietary
HaematoxylinLeica BiosystemsDS9263Proprietary
DAB EnhancerLeica BiosystemsAR9452Proprietary
Washes/BlocksBond Wash (Tris Buffer)Leica BiosystemsAR9590Proprietary
Antibody DiluentLeica BiosystemsAR9352Proprietary
Avidin/Biotin BlockVector LaboratoriesSP-2001Proprietary

Antibody details

Anti-ARID1A is a monospecific rabbit polyclonal generated to a PrEST sequence – PGLGNVAMGPRQHYPYGGPYDRVRTEPGIGPEGNMSTGAPQPNLMPSNPDSGMYSPSRYPPQQQQQQQQRHDSYGNQFSTQGTPSGSPFPSQQTTMYQQQQQNYK (Table 2). The lot number used for all validations was A40072 and for subsequent staining was D81856. A concentration of 1 µg/ml was used for initial validations and 0.5 µg/ml for final runs.

Table 2. Details of primary and secondary antibodies.

AntibodyManufacturerCatalogue NumberRRID
ARID1AAtlas AntibodiesHPA005456AB_1078205
GAPDH (Clone 14C10)Cell Signaling2118AB_561053
Donkey anti-Rabbit BiotinJackson Immunoresearch711-065-152AB_2333077
Goat anti-Rabbit IRDye 680LTLi-Cor Biosciences926-68021AB_10706309
Goat anti-Rabbit IRDye 800CWLi-Cor Biosciences926-32213AB_621848

Donkey anti-rabbit biotin (Jackson Immunoresearch, Table 2) is specific for Rabbit IgG (Heavy and Light chains) and was affinity purified to remove cross-reactions to Bovine, Chicken, Goat, Guinea Pig, Syrian Hamster, Horse, Human, Mouse, Rat and Sheep. All slides were stained with a concentration of 4.8 µg/ml.

Anti-GAPDH was used as a loading control for Western blots and was a rabbit monoclonal (Cell Signaling, Table 2). Detection antibody for the Western blot for ARID1a was Goat anti-rabbit IR Dye 680LT (Li-Cor Biosciences, Table 2) used at a concentration of 0.1 µg/ml and detection of GAPDH was Goat anti-rabbit IR Dye 800CW (Li-Cor Biosciences, Table 2).

Tissue details

All tissues and cell pellets used during the validation were fixed in Neutral Buffered Formaldehyde as specified (Table 3) before being transferred directly to 70% ethanol for no longer than 3 days. Tissue processing was conducted on a Leica ASP300 through graded ethanols before clearing in xylene and impregnation in molten paraffin wax (Fisher). All tissue sections were cut on a Leica rotary microtome at 3 µm.

Table 3. Details of tissue and cell pellet used during the validation.

SpeciesTissue TypeStrain/Cell lineDetailsFixation
Time
MurineUterusC57Bl6Female16 hrs
MurineUterusC57Bl6 KO Arid1afl/fl Female24 hrs
HumanCell PelletES-220 hrs
HumanCell PelletRMG-II20 hrs

Table 4. Staining protocol for ARID1A immunohistochemistry.

Protocol stepsReagentTime
(mins)
Antigen RetrievalER120
Or ER220
Or Enz110
StainingPeroxide Block5
Avidin10
Biotin10
ARID1A15
Donkey anti-
rabbit Biotin
8
SA-HRP8
DAB5
DAB Enhancer10
CounterstainingHaematoxylin5

Experiment details

Western blot. Protein was extracted from the two clear cell carcinoma cell lines using a Tris-EDTA lysis buffer and run on a non-denaturing 3–8% Tris-acetate gel (Life Technologies). Following electrophoresis, the transfer membrane was probed with 0.2 µg/ml of anti-rabbit ARID1A (HPA005456) at 4°C overnight and 0.1 µg/ml anti-GAPDH (14C10) for the same length of time. Detection of the anti-rabbit ARID1A was with Goat anti-rabbit IRDye 680LT (Li-Cor Biosciences) and the GAPDH was with Goat anti-rabbit IRDye 800CW (Li-Cor Biosciences) both at 0.1 µg/ml.

Immunohistochemistry. Slides were deparaffinised and rehydrated on a Leica ST5020 using Xylene (Sigma) for 2 × 10 mins and ethanol (Fisher), 2 × 100% ethanol followed by 1 × 70% ethanol for 5 mins each. Following staining, all slides were dehydrated, cleared and mounted and coverslipped in DPX (Fisher).

The antibody was validated on a Leica BondMax instrument using a Leica Intense R kit to a standardised in-house protocol. All reagents were from Leica as part of the Intense R kit and were conducted at room temperature, unless otherwise specified. All staining steps included individual washes in Leica Bond Wash after each step, as part of the protocol (Table 4). A full protocol for the validated conditions can be found in the supplementary material. In this protocol, the step named “primary” refers to the anti-ARID1a primary antibody.

A slide using the same conditions and retrieval but omitting the primary antibody was used to control for any background staining due to the retrieval and detection steps.

Imaging. All slides were digitised using a Leica Scanscope AT2 at 0.5 µm/pixel resolution. Datasets can be viewed by downloading the Leica Imagescope free viewer at http://www.leicabiosystems.com/pathology-imaging/aperio-epathology/integrate/imagescope/.

Results

To determine the correct cell line to utilise and to confirm the equivocal Western blot data from Human Protein Atlas, the antibody was used to stain a Western blot of two cell lines; ES-2 and RMG-II, both of which are cell lines derived from clear cell carcinoma. It could be demonstrated that the HPA ARID1A antibody showed positive expression in ES-2 cell lines at the expected size of 270 kDa and no staining for RMG-II. The loading control of GAPDH showed that there were no loading issues (Figure 1). Thus, these cell lines were chosen to be grown, formalin fixed and processed into paraffin wax for immunohistochemical validation.

410d4331-cb7c-4eea-ac38-43f5af8a46b1_figure1.gif

Figure 1. Western blot of ES2 and RMG-II clear cell carcinoma cells lines.

ARID1A (red band) can be seen to be present at approximately 270kD in ES2 cell line only. GAPDH at 37kD (green band) represents loading control.

For immunohistochemical validation, ES-2 and RMG-II cell lines were stained using three antigen retrieval conditions; ER1 (Sodium Citrate, pH6), ER2 (Tris/EDTA, pH9) and Enzyme 1 (Proteinase K, 100 µg/ml) at a fixed antibody concentration of 1 µg/ml. The enzyme retrieval demonstrated no nuclear signal for either ES2 or RMG-II cell pellets and was discarded for future work (Figure 2; Dataset a). The ER2 condition did demonstrate significant nuclear staining in the ES2 cell pellet with minimal background staining in the RMG-II cell pellet (Figure 2; Dataset b). However, the staining in the ER1 condition was determined to give the best signal:noise ratio with no background cytoplasmic staining and crisp nuclear staining for the cell pellet (Figure 2; Dataset c). Control slides, omitting the primary antibody, were negative except for the ER2 condition in the RMG-II cell pellet where a weak cytoplasmic background could be seen (Figure 2; Dataset d). Thus there was minimal background inherent in the staining procedure. It was therefore determined that the antibody showed specificity for formalin-fixed paraffin embedded tissues and could be run on murine tissue.

410d4331-cb7c-4eea-ac38-43f5af8a46b1_figure2.gif

Figure 2. ES2 and RMG-II cell lines stained by immunohistochemistry with anti-ARID1A using three antigen retrieval conditions, ER1, ER2 and Enzyme 1.

NPA denotes No Primary antibody control and represents the ER2 condition. Bar = 200 µm.

Murine uterine tissue was used as positive control tissue samples, given the literature data on cell lines and endometrial tissue. The ER1 condition at 1 µg/ml demonstrated clean nuclear staining in the uterine epithelial compartment as well as nuclear staining of stromal cells. However, the nuclear staining in the stroma was not universal and distinct negative nuclei could be seen (Figure 3; Dataset e). There was no cytoplasmic or extracellular stromal background staining present and the antibody titrated successfully losing the intensity of staining, as expected (Dataset e). Following this, a concentration of 0.5 µg/ml was used for future preparations which provided clear and consistent staining in repeated batches using a different antibody lot (Dataset f).

410d4331-cb7c-4eea-ac38-43f5af8a46b1_figure3.gif

Figure 3.

Murine uterine tissue stained by immunohistochemistry with anti-ARID1A using the ER1 condition and a concentration of 1 µg/ml demonstrating clear nuclear staining of the epithelial compartment (E) and negative nuclei in the stromal compartment (S). Bar = 100 µm.

Finally, when applied to a genetically engineered, tamoxifen-induced, Arid1a knockout mouse model, the staining in the uterine epithelium could be completely abrogated (Arrow, Figure 4b) when compared to the same area in a wild-type animal (Arrow, Figure 4a), while not affecting the staining in the stromal compartment.

410d4331-cb7c-4eea-ac38-43f5af8a46b1_figure4.gif

Figure 4.

Murine uterine tissue stained by immunohistochemistry with anti-ARID1A demonstrating nuclear staining in wild-type mice (A) but loss of epithelial staining after ARID1A knock-out in Arid1afl/fl mice (B). Bar = 100 μm.

Dataset 1.Whole slide images from antibody validation of HPA005456 for immunohistochemistry.
Detailed legends for each dataset (Datasets a–f) can be found in the text file provided

Conclusions

It is clear from the use of ES2 and RMG-II cell lines that the Atlas Antibodies ARID1A antibody is specific for ARID1A in both Western blots and formalin-fixed paraffin embedded preparations of human origin and, coupled with the literature evidence, that it is validated in human tissue.

The staining pattern when applied to murine uterus showing a clear nuclear pattern, is again consistent with the literature on this protein. Crucially, however, when stained on an ARID1a knockout mouse model, the staining could be almost completely abrogated in the epithelial compartment and thus when taken in combination, it is clear that the anti-human ARID1a antibody is cross-reactive with murine tissue and can be used for this purpose.

Data availability

F1000Research: Dataset 1. Whole slide images from antibody validation of HPA005456 for immunohistochemistry, 10.5256/f1000research.5514.d3676110

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how to cite this article
Howat W, Miller J and Gounaris I. Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:244 (https://doi.org/10.12688/f1000research.5514.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 15 Oct 2014
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Reviewer Report 02 Dec 2014
Stephen McQuaid, Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK 
Approved with Reservations
VIEWS 32
This article, by Howatt et al. validates an antibody to ARID1A in murine FFPE samples, is a good example of the correct validations procedures that must be met when validating an antibody for use in tissue sections. Issues are relatively ... Continue reading
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CITE
HOW TO CITE THIS REPORT
McQuaid S. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:244 (https://doi.org/10.5256/f1000research.5886.r6848)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 06 Jan 2015
    Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK
    06 Jan 2015
    Author Response
    Dear Dr McQuaid,

    Thank you for your comments which we agree with and have taken on board. Specifically:
    • All of the validations conducted at the Histopathology/ISH facility start with a dilution of
    ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 06 Jan 2015
    Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK
    06 Jan 2015
    Author Response
    Dear Dr McQuaid,

    Thank you for your comments which we agree with and have taken on board. Specifically:
    • All of the validations conducted at the Histopathology/ISH facility start with a dilution of
    ... Continue reading
Views
30
Cite
Reviewer Report 21 Nov 2014
Andrew D. Chalmers, Department of Biology and Biochemistry, University of Bath, Bath, UK 
Approved with Reservations
VIEWS 30
The manuscript by Will Howat and colleagues validates an anti-ARID1A antibody and provides a good example of an antibody validation paper. In particular the use of negative cell lines and knockout mouse tissue demonstrate the extent of specificity of the ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Chalmers AD. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:244 (https://doi.org/10.5256/f1000research.5886.r6651)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 06 Jan 2015
    Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK
    06 Jan 2015
    Author Response
    Dear Dr Chalmers,

    Many thanks for your comments which we agree with and have taken on board. Specifically:
    • We have modified the title to read "Application of anti-ARID1a antibody..."
       
    • We have BLAST searched
    ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 06 Jan 2015
    Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK
    06 Jan 2015
    Author Response
    Dear Dr Chalmers,

    Many thanks for your comments which we agree with and have taken on board. Specifically:
    • We have modified the title to read "Application of anti-ARID1a antibody..."
       
    • We have BLAST searched
    ... Continue reading

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 15 Oct 2014
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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