Biotinylated swine anti rabbit igg

Cerebellum sections were dewaxed, rehydrated, and placed in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. After rinsing in PBS (pH 7.4) with 0.1% Tween-20 (PBST), the sections were blocked with 5% BSA and 5% normal goat serum (NGS) in 0.1 M PBS (pH 7.4) for 60 min at room temperature, followed by overnight incubation with PSAP-Ab (1 μg/mL), anti-GPR37 (1 μg/mL), or anti-GPR37L1 (2 μg/mL) at 4 °C.
For IHC, biotinylated swine anti-rabbit IgG (1:500; Dako, Glostrup, Denmark) was then added and incubated for 1 h at 32 °C. Antibody binding was detected with avidin-biotin-peroxidase complex (1:100; Vector Laboratories, Burlingame, CA, U.S.A.) using a DAB Peroxidase Substrate Kit (1:100; Vector Laboratories, Burlingame, CA, U.S.A.) for approximately 5 min. After washing with distilled water, the sections were mounted and examined under a microscope.
For IF, after washing with PBS, the sections were treated for 30 min at 32 °C with fluorescent anti-rabbit IgG (1:500, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI, 1:1000). The sections were then washed with PBST, mounted with Vectashield (1:100; Vector Laboratories, Burlingame, CA, U.S.A.), and examined using a confocal laser scanning microscope.
To ensure accurate and specific staining, normal rabbit IgG (1 μg/mL) was used as a negative control. Nonspecific staining was not observed.