Anti mouse cd31 antibody

Mice were sacrificed at 1–7 days after wounding, and skin tissues around wound edge were harvested and embedded in paraffin. Frozen sections were prepared by overnight 4% PFA incubation followed by sucrose dehydration and OCT embedding. 5-μmthick sections were stained with hematoxylin-eosin (HE), and Masson’s trichrome staining. To determine the Capillary density, wound tissue sections were stained with anti-mouse CD31 antibody (BD Biosciences) followed by biotinylated anti-mouse IgG antibody (Vector Laboratories). For other immunohistochemical analysis, skin sections were incubated with primary antibodies against Atox1, Mac-3 (BD Biosciences) and cyclin D1 (Abcam). This was followed by incubation with biotin-conjugated secondary antibody (Vector Laboratories) Next, we used R.T.U. Vectorstain Elite (Vector Laboratories) followed by DAB visualization (Vector Laboratories). Immunofluorescence staining was performed with primary antibodies against Atox1, CD31 (BD Biosciences), or Mac-3 (BD Biosciences). Secondary antibodies were Alexa Fluor 488 or 546-conjugated goat anti-rabbit IgG and goat anti mouse IgG (Invitrogen). In each experiment, TO-PRO-3 (Invitrogen) was used for nuclear counter staining. Images were captured by Axio scope microscope (Zeiss) or confocal microscopy (Zeiss) and processed by AxioVision 4.8 or LSM510 software (Zeiss).

Six-week-old male KSN nude mice were anesthetized with pentobarbital, and the right common iliac artery was resected. After surgery, amnion MSCs (1×10⁶ cells/50 µL PBS), chorion MSCs (1×10⁶ cells/50 µL PBS), or PBS (50 µL PBS) was injected into the ischemic muscle with a 30-gauge needle at five different sites (n = 15 in each group). A laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, Devon, UK) was used to measure serial hindlimb blood flow for 7 days, as previously described [12] (link).
Five and seven days after MSC transplantation, ischemic hindlimb tissues were obtained and snap-frozen. Frozen tissue sections were stained with anti-mouse CD31 antibody (BD Biosciences) to detect capillary endothelial cells. Ten fields were randomly selected to count the number of capillaries. The adjusted capillary number per muscle fiber was used to compare the differences in capillary density between the three groups.

For cryosections, mice were euthanized and perfused through the left ventricle with saline and 4% paraformaldehyde, limbs were fixed in 4% paraformaldehyde (PFA) overnight and incubated with 30% sucrose, and adductor and gastrocnemius muscles were embedded in OCT compound (Sakura Finetek). For paraffin sections, we performed methanol fixation or PFA fixation with decalcification by Immunocal (Decal Chemical Corp.). Capillary density in the ischemic muscles was determined in 7 µm cryosections or in 5 µm methanol fixed paraffin sections that were stained with anti-mouse CD31 antibody (BD)(1:300). For immunohistochemistry, we used R.T.U. Vectorstain Elite (Vector Laboratories) followed by DAB visualization (Vector Laboratories). Images were captured by Axio scope microscope (Zeiss) or confocal microscopy (Zeiss) and processed by AxioVision 4.8 or LSM510 or ZEN 2.3 software (Zeiss).