Immunohistochemical Analysis of Caveolin-1 in Adipose Tissue

Paraffin-embedded sections (4 μm thick) of subcutaneous AT were deparaffinized in xylene and rehydrated through descending grades of ethanol (100%, 95%, and 75%) to water. Antigen retrieval was performed by placing slides in a target retrieval solution (pH 6.0; Dako, Glostrup, Denmark) in a pressure cooker, boiling for 8 min, and cooling for 15 min. After washing in PBS, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min, and nonspecific antibody binding was blocked with 5% fat-free milk for 1 h, followed by 1% bovine serum albumin solution for 1 h. The slides were incubated at room temperature overnight with primary antibody (1:100 dilution of rabbit polyclonal anti-Caveolin-1 antibody; Cell Signaling Technology #3238s). After washing with PBS (0.5% Tween), slides were incubated for 1 h with a secondary antibody, namely, goat anti-rabbit conjugated with horseradish peroxidase (HRP) polymer chain Dako EnVision Kit from (Dako, Glostrup, Denmark) and color was developed using 3,3′-diaminobenzidine (DAB) chromogen substrate. Specimens were washed, counterstained, dehydrated, cleared, and mounted, as described elsewhere [25 (link)]. For analysis, digital photomicrographs of the entire AT sections (20X; PannoramicScan, 3DHistech, Hungary) were used to quantify the immunohistochemical staining using ImageJ software (NIH, Bethesda, MD, USA).