Super sensitive detection kit

Four micrometer sections of formalin-fixed and paraffin-embedded conjunctival samples were prepared. Immunohistochemical staining was performed using a primary clone 1A4 antihuman mouse monoclonal α-SMA antibody, prediluted to 1:50 dilution in 1% bovine serum albumin (Cat. #M0851, Dako, Carpinteria, CA, U.S.A.). Avidin–Biotin immunoperoxidase complex technique was used, by applying a super sensitive detection kit (Biogenex, CA, U.S.A.) according to the manufacturer instructions to detect the bound antibody. The prepared tissue sections were fixed on poly-L-lysine coated slides overnight at 37°C. They were deparaffinized and rehydrated through graded alcohol washes, then the sections were heated in a microwave oven in 10 mM citrate buffer (pH 6.0) for 10 minutes. After the blocking of endogenous peroxidase and incubation in Protein Block Serum-Free Solution (DakoCytomation, Glostrup, Denmark) for 20 minutes, the sections were incubated with α-SMA antibody at room temperature. Biotinylated antimouse immunoglobulin and streptavidin conjugated to horseradish peroxidase were then added, and then chromogen was used to form an insoluble brown product. Finally, the sections were counterstained with hematoxylin and mounted. Vascular smooth muscle was used as an internal control.