Immunohistochemical detection of steroid binding in rabbit cornea

An indirect immunoperoxidase technique was used to demonstrate progesterone binding in rabbit cornea as a measure of steroid absorption into corneal tissues. Tissue preparation prior to immune staining was prepared by both a pre-fixed, paraffin-embedding method and a post-fixed, cryostat sectioning method. Rabbit antiprogesterone antiserum and peroxidase conjugated goat antirabbit antiserum were used as a primary and secondary antibody respectively. The immune staining of the antibody-treated sections was then developed in a solution of 3-3' diaminobenzidine-hydrogen peroxide system. The resulting, brown colored, end-products at the site of reaction indicate the location of bound steroid in the sections. Paraffin-embedded sections showed negative staining in both steroid treated and untreated control corneas while fixed frozen sections showed only positive staining for the treated corneas. The results suggest the preservation of the immunogenicity in the treated frozen sections. The negative result observed in the paraffin-embedded sections may be attributed to the loss of steroid from the tissues during the dehydration and paraffin embedding processes. It was found that the immunoreactive progesterone localized mainly in the epithelial cells and the bottom portion of the stroma. Prior application with an excess of triamcinolone acetonide, to the treated corneas gave negative results indicating the reversible and competitive nature of the binding.