Avidin biotin peroxidase complex solution abc

Sections (5 μm-thick) of kidney tissues were deparaffinized, rehydrated and immersed in 10 mM citric acid (pH 6.0) for antigen retrieval. The sections were incubated with primary antibodies against Grp78 (1:100), CHOP (1:50), TGF-β1 (1:100) and TSP-1 (1:100) overnight at 4°C, followed by incubating with biotinylated goat anti-rabbit IgG (1:200, # BA1003; Boster Biological Technology, Wuhan, China) in phosphate-buffered saline (PBS) for 2 h at room temperature. After a further incubation in avidin-biotin-peroxidase complex solution (ABC, 1:100, Boster Biological Technology, Wuhan, China) for 2 h at room temperature, the respective antigens were visualized with 0.05% 3,3ʹ-diaminobenzidine (DAB; #D5905; Sigma-Aldrich) with H&E counterstaining. Each section was scanned using a Panoramic MIDI (3DHISTECH, Hungary). IHC staining was scored independently by two pathologists [percentage of positive cells: five categories: 0 (<10%), 1 (10–25%), 2 (25–50%), 3 (50–75%), and 4 (75–100%); intensity: four categories (from low to high): 0, 1, 2, and 3; and a final IHC staining score (intensity score × percentage score)] was determined.18 (link)

For immunohistochemistry, the separated sections were washed in 0.01 M PBS containing 3% Triton X-100 (pH 7.4, PBS-T), then inserted in X10 normal goat serum in PBS for 45 min at 37°C, and then incubated at 4°C with monoclonal anti-GAP-43 (1:200, Abcam) or anti-MKP-1 (1:200, Monoclone MKP-1 antibody, Santa Cruz Biotechnology, Lot# D0904, USA) in PBS for overnight, washed in PBS (3 × 5 min), incubated in biotinylated horse anti-mouse IgG (1:200, Boster) in PBS for 2 h at room temperature, washed in PBS (3 × 5 min), incubated in avidin-biotin-peroxidase complex solution (ABC, 1:100, Boster) for 2 h, then washed again in PBS (3 × 5 min). The nucleus stained by incubating the tissue by DAPI for 15 min.