Biotinylated anti goat igg secondary antibody

Paraffin-embedded tissue blocks were cut into serial sections (4 µm) and the expression of Ki-67, Bax, Bcl-2 and cyclin D1 were determined by immunohistochemistry staining. Briefly, the sections were deparaffinized using citric acid buffer (pH 6.0), and then the slides were treated with 3% hydrogen peroxide to block endogenous peroxidase activity before incubation with Ki-67 (GB13030-2, 1:1,000 diluted in 1% BSA), Bcl-2 (GB12008-1, 1:400), Bax (GB11007, 1:300) and cyclin D1 (GB GB13079, 1:200) primary antibodies. The slides were then incubated with 5 µg/ml biotinylated anti-goat IgG secondary antibody (Dako, Carpinteria, CA, USA) for 30 min at room temperature. After washing, slides were stained with 3,3-diaminobenzidin (DAB; Dako), and then counterstained with hematoxylin, dehydrated and mounted with a coverslip. For hematoxylin and eosin (H&E) staining, sections were deparaffinized with xylene for 10 min and then rehydrated. The sections were stained with hematoxylin for 10 min and then stained with eosin for 1 min. The sections were dehydrated and mounted. All images were captured using an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Formalin-fixed tumor tissues were embedded in paraffin and cut into 4-µm sections. The sections were then stained with H&E for pathological evaluation. The rest of the paraffin blocks were cut into 4-µm sections. The sections were deparaffinized using citric acid buffer (pH 6.0) and incubated for 8 min at 100°C. The slides were treated with 3% hydrogen peroxide to block endogenous peroxidase activity and then incubated with 1% bovine serum albumin (BSA) for 25 min. Next, the slides were incubated overnight at 4°C with anti-human primary antibodies: Bcl-2 (GB12008, 1/100 diluted in 1% BSA), Ki67 (GB13030-2, 1/1000 diluted in 1% BSA), PCNA (GB11010, 1/500 diluted in 1% BSA). All primary antibodies were obtained from Wuhan Goodbio Technology Co., Ltd. (Wuhan, China). The slides were then incubated with 5 µg/ml biotinylated anti-goat IgG secondary antibody (Dako, Carpinteria, CA, USA) for 50 min at room temperature. After washing, slides were stained with 3,3-diaminobenzidine (DAB) (Dako), washed and counter-stained with hematoxylin, dehydrated, and then mounted with a coverslip. All images were captured using an inverted fluorescence microscope (Nikon, Japan).