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

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 lesions^1–4. 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

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 and have been previously demonstrated as ARID1a wild-type and mutated, respectively⁹. 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.

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.

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 3A; 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). A No Primary antibody control (NPA) showed no staining in the epithelial or nuclear compartment (Figure 3B; Dataset e).

Figure 3.

A) Murine uterine tissue stained by immunohistochemistry with anti-ARID1A using the ER1 condition and a concentration of 1 ug/ml demonstrating clear nuclear staining of the epithelial compartment (E) and negative nuclei in the stromal compartment (S). B) No primary antibody control of a similar area of epithelium/stroma. Bar = 100 µm.

Finally, when applied to a genetically engineered, tamoxifen-induced, Arid1a knockout mouse model, the staining in the uterine epithelium could be almost completely abrogated (Arrow, Figure 4b) when compared to the same area in a wild-type animal (Arrow, Figure 4a) with a small focal area of epithelial staining still present. There was no effect of the KO on the staining in the stromal compartment.

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 Arid1a^fl/fl mice (B). Bar = 100 μm.

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, where there is a high level of sequence homology between the two species, is again consistent with the literature on this protein. When stained on an ARID1a knockout mouse model, the staining could be almost completely abrogated in the epithelial compartment but not in the stroma. Knockout mice generated in this manner are almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (Tamoxifen) penetration or failure of the ligand to induce recombination, thus explaining the small focal area of epithelial staining. The difference in staining in the two compartments is also likely related to the same effect, as all other controls, such as omission of primary antibody remained negative. Thus, given the overwhelming data from other sources, it is likely the stromal staining reflects continuing Arid1a expression in this specific model system.

Therefore, in conclusion 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 - Version 2, 10.5256/f1000research.5514.d41579¹⁰