Immunohistochemistry for GR and HO-1

Immunohistochemical techniques were performed as previously described [6 (link),11 (link)]. Briefly, immunohistochemistry was done using the streptavidin-biotin-peroxidase complex system LSAB + kit, horseradish peroxidase (DAKO, Santa Clara, CA, USA). Endogenous peroxide activity was quenched using hydrogen peroxide in distilled water (3%). Antigen retrieval was done by microwaving. Tissue slides were incubated overnight with GR and HO-1 primary antibodies. This was followed by sequential incubations with biotinylated antibody and peroxidase-labelled streptavidin complex. The peroxidase reaction was conducted, under microscope, using 3,3′-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin and analyzed by standard light microscopy. Negative control slides were prepared by substituting primary antiserum with PBS. For semiquantitative analysis, the degree of staining was scored as high, moderate, low, or not detectable (3+, 2+, 1+, and 0, respectively); staining localization was also recorded. We considered positive expression when more than 10% cells exhibited positive staining.