Bovine serum albumin bsa

IHC was performed according to the manufacturer's protocol. To retrieve antigens in the skin tissues, the deparaffinized sections were boiled in 10 mM sodium citrate buffer for 30 min. Endogenous peroxidase was subsequently blocked by incubation in 3% H₂O₂ for 30 min and slides were blocked with 5% normal goat serum (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in phosphate-buffered saline (PBS) containing 5% fetal bovine serum (Corning, Inc., Corning, NY, USA), 2% bovine serum albumin (BSA; Vector Laboratories, Burlingame, CA, USA) and 0.1% Triton X-100 for 1 h. Following washing in PBS three times, the sections were incubated overnight with an antibody against biotin anti-mouse CD4 (cat. no. 100403; BioLegend, Inc., San Diego, CA, USA), diluted 1:100 in normal goat serum, in a humidified chamber. After washing with PBS, the biotinylated goat anti-mouse IgG (cat. no. BA-9200; diluted 1:200 in TBST; Vector Laboratories) was applied for 2 h at room temperature. The slides were subsequently incubated with an Avidin/Biotinylated Enzyme Complex kit (Vector Laboratories) for 1 h, followed by the substrate chromogen (3,3-diaminobenzidine; Dako, Glostrup, Denmark), and counterstained by hematoxylin staining. CD4 staining was visualized using LAS (magnification, ×200).