Improvement of antibody staining for limbal-corneal tissue by using Tris/EDTA buffer for epitope retrieval

Abstract

Purpose: The focus of our research is the identification of the limbal stem cells and their niche cells. Until now a set of positive and negative markers for limbal stem and progenitor cells has been described. Here we re-evaluated the expression pattern of established markers immunohistochemically under different buffer conditions in order to identify a staining protocol for stem cell specific markers for further studies.

Methods: Limbal cell cultures were grown out from a biopsy on amniotic membrane in supplemental hormonal epithelial medium. These cultures and paraffin embedded limbal-corneal tissue slices were subjected to immunofluorescence staining with a set of markers for limbal stem cells and corneal epithelial differentiation (K12, K15, K19, connexin 43, p63, ABCG2 and ABCB5). For epitope retrieval we compared the widely used citrate buffer (pH 6.0) with a Tris/EDTA buffer (pH 9.0).

Results: K15 was expressed in the basement membrane of the limbus and the transitional zone towards the cornea. It was also expressed in a small subset of cultured cells. K12 and connexin 43 are mostly expressed in the cornea and the suprabasal layers of the limbus. A large amount of cells grown on amniotic membrane were also positive for these two differentiation markers. There was also a large number of ABCB5 and p63 positive cells in the cultures on amniotic membrane. Epitope retrieval performed better with the Tris/EDTA buffer for 6 out of 7 of the tested antibodies (K12, K15, K19, connexin 43, ABCG2 and ABCB5). For p63 Tris/EDTA buffer showed a wrong staining pattern so we recommend citrate buffer for this marker the other way around is it with ABCG2. Noticeable the epitope retrieval with Tris/EDTA buffer (pH 9.0) enabled a much stronger dilution of the antibody compared to the citrate buffer and produced a very specific staining pattern.

Conclusions: Using the Tris/EDTA buffer for epitope retrieval offers the advantage of using a higher dilution of the antibody compared to the citrate buffer and thus saves material and produces a specific staining at the same time. Because of the wrong staining pattern of some markers with one of the buffers we recommend to test both for each new antibody.