Streptavidin conjugated horseradish peroxidase hrp

Paraffin sections (4 μm) from samples were deparaffinized in 100% xylene and re-hydrated in descending ethanol series and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and non-specific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum, followed by incubation with rabbit anti-human ENO1 antibody (1: 150) (48 kDa, Proteintech, USA) overnight at 4°C. After washing, the sections were incubated with biotin-labeled rabbit anti-goat antibody for 10 min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin, Fuzhou, China). The peroxidase reaction was developed using 3,3-diaminobenzidine (DAB) chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope.

Paraffin sections (4 mm) were deparaffinized in 100% xylene and re-hydrated in descending ethanol series and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and non-specific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum. The sections were then incubated with primary antibodies, including HIF-1 α (1:50; Novus Biologicals, USA), PLOD2 (1:100;Proteintech, USA), E-cadherin (1:100;Proteintech, USA), β-catenin (1:100; Proteintech, USA), and snail (1:100; Abcam, USA), at 4°C overnight. After washing, the sections were followed by incubation with biotin-labeled rabbit anti-goat antibody for 15 min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin, Fuzhou, China). The peroxidase reaction was developed using 3,3-diaminobenzidine (DAB) chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope equipped with a digital camera (Nikon, Japan).