3 3 diaminobenzidine tetrahydrochloride dab

For NRP2 immunostaining of human primary tumors, freshly frozen samples of human primary breast tumors were sectioned, fixed in acetone, and immunostained with anti-NRP2 antibody, anti Ki-67 antibody (Abcam, Cambridge, MA, USA). The samples were incubated in biotinylated anti-rabbit secondary antibody (Beyotime, Shanghai, China) followed by 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Beyotime). The slides were counterstained with hematoxylin.

Immunohistochemistry (IHC) was performed as previously described [36 (link)]. The ovarian tissues of mice were fixed in 4% formaldehyde and paraffin-embedded using standard procedures. First, consecutive 4-µm sections were cut, deparaffinized with xylenes, rehydrated, and retrieved the antigen in sodium citrate solution (pH 6.0) for 20 min. Then the slides were treated with 3% hydrogen peroxide to quench the endogenous peroxidase and blocked with 1% bovine serum albumin for 30 min to block the nonspecific binding. Next, tissue sections were incubated with primary antibody anti-FTO (Abcam, Cambridge, ab126605, USA; 1:150) overnight at 4 ℃. Finally, wash the slides with PBS three times and incubate the slides with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Santa, Cruze) for another 30 min. Finally, a 3, 3-diaminobenzidine tetrahydrochloride (DAB) (Beyotime, Wuhan, China) substrate kit was applied to detect peroxidase reactivity. According to the manufacturer instructions, we prepared DAB peroxidase substrate in 5 ml ddH₂O in a glass vial. Then, drop the DAB substrate on top of the slides and watch the brown staining. Dip slides into ice plus tap water to stop the reaction and rinse under cold tap water for 5 min.