Diaminobenzidine peroxidase dab substrate

Specimens were harvested and fixed overnight in 10% formalin/phosphate-buffered saline and embedded in paraffin blocks. The proliferative capacity of the small intestine and apoptosis were determined by immunohistochemistry for BrdU (Abcam, ab6326), Ki67 (Abcam, ab15580) and active caspase-3 (Cell Signaling Technologies, 9579S). The cellular composition of the intestine was determined using antibodies for Dclk1 (Abcam, ab37994), chromogranin A (Abcam, ab15160), Muc2 (Santa Cruz Biotech, sc-15334), lysozyme (Dako, A0099), and synaptophysin (Abcam ab32127). Biotinylated secondary antibodies were applied at 1:1000 dilutions and visualized by using diaminobenzidine peroxidase (DAB) substrate (Invitrogen, Carlsbad, CA). Slides were counterstained with hematoxylin. For immunofluorescent staining, secondary antibodies were applied at 1:500 dilution and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Femur sections were pretreated with 0.1% Tween in PBS for 30 min. Tissue sections were blocked in 5.0% BSA for 2 h before incubation with 5 μg/ml rabbit polyclonal antibody against OPG or RANKL overnight at 4 ℃. Then, the tissue sections were washed in TBST and incubated with HRP-conjugated secondary antibodies (Zen-Bioscience Company, China). The color was developed using diaminobenzidine (DAB) peroxidase substrate (Invitrogen, UK) for 2 min before counterstaining with hematoxylin (Solarbio, Beijing, China). Images were analyzed with the ImageJ software (NIH, MD) for quantifying the intensity of the reaction (Mean density = IOD Sum/Area Sum).