Primary antibody against cd31 rat anti mouse

On days 7, 14, and 30, mice were executed to harvest heart samples. Then, the heart tissues were fixed with 4% formaldehyde and embedded in paraffin. Then, paraffin tissues were cut into 6–8-μm-thick sections, which were stained with Masson’s staining and hematoxylin and eosin (HE). The ratio of collagen area to the total left ventricular area was taken as the collagen content. Image J software was used to measure the area of collagen and LV.
For immunofluorescent staining, the heart tissues were embedded into OCT compound (Sakura Finetek, Tokyo, Japan). Then, the fixed tissue was cut into 6–8-μm-thick sections. The sections were incubated with primary antibody against CD31 (rat anti-mouse; Abcam, Cambridge, MA) over night, and then, AlexaFluor 594 goat anti-rat IgG (Invitrogen, Grand Island, NY) was dripped. Cell nuclei were counterstained with 6-diamidino-2-phenylindole (Southern Biotech, Birmingham, AL). The total amount of capillaries in the infarct area were determined in three random fields.

For angiogenesis analysis, animals were euthanized and the muscle tissues were embedded into OCT compound (Sakura Finetek, Japan). Samples were cut into 6-μm-thick sections for immunofluorescence staining. The sections were incubated with primary antibody against CD31 (rat anti-mouse, Abcam, Cambridge, MA) overnight at 4 °C, then incubated with Alexa Fluor 594 goat anti-rat IgG (Invitrogen, Grand Island, NY) and counterstained with 6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL). The number of capillary vessels was randomly counted in ten selected areas with a fluorescence microscope (× 200). Hematoxylin-eosin (HE) staining and Masson’s trichrome staining were performed to investigate the therapeutic effects of IGF-1C hydrogel and hP-MSCs on day 21.