Il 12p40

Immunohistochemical analysis was conducted as described in our previous study [9] (link). Briefly, after getting rid of paraffin from tissues on the slides in order to eliminate endogenous peroxidase they were immersed in 3% hydrogen peroxide in methanol for 10 min. Sodium citrate buffer (0.1 M) was used for the antigen retrieval. In order to prevent non-specific binding normal horse serum was used, and then they were bounded with primary antibodies (diluted 1:100 to 1:200) including IFN-γ (Santa Cruz, sc-74104), IL-12p40 (Santa Cruz, sc-57258), IL-4 (Santa Cruz, sc-73318), IL-5 (Santa Cruz, sc-7887), IL-13 (Santa Cruz, sc-1776), TNF-α (BioVision, 3053R-100, Milpitas, CA, USA), and IL-6 (Santa Cruz, sc-1265) antibodies at 4 • C for 1 h. Using biotinylated secondary antibody, all slides were incubated for 10 min and reacted with streptavidin peroxidase complex for 5 min (Vector Laboratories Universal Quick Kit, Burlingame, Canada). Signals were obtained using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution and they were stained with Mayer's hematoxylin. The results were obtained by an Axioscope A1 (Carl Zeiss) and to measure the positive cells we counted them in five randomly selected non-overlapping fields (×200 magnification) of three separately.