Rabbit anti mouse cd31 antibody

Immunohistochemistry of tissue sections with rabbit anti-mouse CD31 antibody (Santa Cruz Biotechnology, USA) was performed at 7 days to detect angiogenesis in the wound after intervention. Sections were deparaffinized, rehydrated, heated in a microwave oven twice for antigen recovery, treated with 3% H₂O₂ and then incubated with 5% goat serum albumin. Then, the sections were incubated overnight at 4 °C with primary rabbit anti-mouse CD31 antibody (1:75, Santa Cruz Biotechnology), followed by a 1 h incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:200, Abcam). After being counterstained with hematoxylin, the slides were assessed with a fluorescence microscope (40FL Axioskop, Zeiss).

Bilateral gastrocnemius and soleus muscles were dissected and snap-frozen in a bath of liquid nitrogen on post-operative day 21. Muscles were sectioned on a cryostat to a thickness of 7 μm and fixed in 100% methanol and H₂O₂. Immunohistostaining was performed with rabbit anti-mouse CD31 antibody (Santa Cruz Biotechnology) and an anti-rabbit Ig horseradish peroxidase detection kit (Nichirei biosciences, Chuo, Tokyo, Japan). The capillary count was performed under light microscopy (magnification ×200). The number of capillaries per muscle fiber was measured in 5 randomly chosen fields from three different sections in each tissue block [60 (link)]. For demonstrate representative morphology of ischemic muscles, sections were stained with Hematoxylin and Eosin (H & E).

Mouse tumors were formalin-fixed, paraffin embedded and 6 μm sections were cut for immunostaining. Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol baths, blocked in 5% normal goat serum (Dako) and subsequently incubated either with rabbit anti-mouse CD31 antibody (1:500)(Santa Cruz), with rabbit anti-mouse Ki-67 antibody (1:300)(Abcam), or with rabbit anti-mouse OGT antibody (1:500)(Cell Signaling). Next, sections were incubated with 0.3% H₂O₂/PBS, followed by polyHRP goat anti-rabbit IgG incubation (Bright Vision). Finally, antibody binding was visualized with 3,3′-diaminobenzidine (DAB).