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A.E. Gallagher, J.J. Hackett, C.C. Chan, R.B. Nussenblatt, Z.Q. Li; Application of Antigen Retrieval and Immunohistochemistry to Study the Expression of GITRL in Normal and Diseased Ocular Tissue . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5113. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine GITRL (glucocorticoid–induced TNF–related receptor ligand) expression differences between normal ocular tissue and ocular tissue affected by inflammatory disease. Using frozen sections of normal human ocular tissue, we have shown GITRL antibody (Ab) staining on the inner segment of photoreceptors (Kim et al, IOVS 2004), but GITRL expression has not yet been shown in paraffin embedded diseased human ocular specimens. This study explored GITRL expression in such tissue. Methods:Antigen retrieval and the avidin–biotin complex immunohistochemical method were performed on formalin fixed, paraffin embedded normal and diseased human ocular tissue to examine GITRL expression. Throughout the study, tissue sections were tested with and without antigen retrieval to evaluate its enhancing effects on Ab staining. The GFAP Ab was used as a control to stain the glial cell layer of the retina in normal ocular tissue. GITRL expression in the inner segment of normal photoreceptors was then explored using a GITRL goat polyclonal Ab (C–18, Santa Cruz Biotechnology), raised against a peptide mapping near the carboxy terminal of human originated GITRL. To confirm the specificity of this GITRL Ab, a peptide block (C–18 P) was performed. GITRL expression was then examined using archived, paraffin embedded patient tissue samples with early and late stage uveitis. Results: As a control, the GFAP Ab was used to successfully stain the glial cell layer of the retina in normal ocular tissue. The C–18 GITRL Ab stained specific to the inner segment of normal photoreceptors. The specificity of the GITRL Ab was confirmed with a peptide block, which abolished GITRL expression in the photoreceptor layer. GITRL expression in the inner segment of photoreceptors in uveitis–ridden ocular tissue patient samples was detected using the C–18 GIRTL Ab. In all cases, while antigen retrieval dramatically enhanced Ab staining, the integrity of the paraffin embedded tissue could be compromised due to intense heat. Conclusions:This study presents the prospect of using the C–18 GITRL Ab to examine the varying GITRL expression in diseased ocular tissue, depending on the disease and its stage. The specificity of GITRL expression in the inner segment of photoreceptors may demonstrate a potential neuroprotective effect in the course of autoimmune disease. Further studies to evaluate differences in GITRL expression between various ocular diseases, as well as various stages of a disease, will provide more insight into this important molecule.
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