En vision k5007

Immunohistochemical staining of KLHL22 was performed using the standard EnVision procedure. First, we sequentially cut the paraffin-embedded tissue blocks into 3 µm thick sections and then dried and deparaffinized the slides with the tissue flakes in xylene. Next, the slides were rehydrated through graded alcohol and immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. After that, the antigen was retrieved by pressure cooking for 3 min in an ethylenediaminetetraacetic acid pH 9.0 antigen retrieval solution. To reduce nonspecific reactions, the slides were incubated with 5% bovine serum albumin for 15 min, after which they were incubated with the mouse anti-KLHL22 monoclonal antibody (1:500 dilution; abs132394; Absin Biotechnology Company, Shanghai, China) for 50 min at 37 ℃. The slides were then incubated with the secondary antibody (Envision, k5007; Dako, Denmark) for 30 min at 37 ℃, followed by staining with 3,3-diaminobenzidine. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. For the negative control, the primary antibody was replaced with normal mouse immunoglobulin G (IgG).