Mouse anti cd31

Paraffin slices of the aortas were deparaffinized, rehydrated, and incubated in goat serum for 1 h at 37 °C. Rabbit anti-p16 (1:300, Affinity Biosciences, Changzhou, China), anti-P21 (1:300, Affinity Biosciences, Changzhou, China), anti-P53 (1:300, Affinity Biosciences, Changzhou, China), anti-SIRT1 (1:100, Boster, Wuhan, China), anti-Nrf2 (1:150, Abclonal, Wuhan, China), anti-HO-1 (1:100, Abclonal, Wuhan, China), anti-IL-6 (1:300, Servicebio, Wuhan, China), anti-TNF-α (1:200, Affinity Biosciences, Changzhou, China), anti-NF-κB (1:200, Affinity Biosciences, Changzhou, China), and mouse anti-CD31 (1:300, Servicebio, Wuhan, China) antibodies were first added and incubated overnight to determine the contents of these proteins in the aortas at 4 °C. After that, FITC-labelled anti-rabbit and anti-mouse IgG antibodies were used as secondary antibodies (Servicebio, Wuhan, China) for detection. One hour later, DAPI was used for nuclear counterstaining. Finally, images were obtained with a fluorescence microscope (Leica, Wetzlar, Germany). ImageJ software was used to quantify the results.

At 7 days after stroke, MCAO rats in each group were perfused with ice-cold PBS followed by 4% phosphate-buffered paraformaldehyde. IF was performed as described previously^{19 (link)}
. The primary antibodies were: Mouse anti-CD31 (GB12063, Servicebio, China), rat anti-von-Willebrand-Factor(anti-vWF) (GB11020, Servicebio, China), The secondary antibodies used were: Cy3-labeled goat anti-mouse IgG (GB21301, Servicebio, China), FITC-labeled goat anti-rabbit IgG (GB22303l, Servicebio, China).
For quantification, three randomly selected high-power fields (HPFs; 400× or 200× for IF study) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was calculated.