Alexa fluor 488 labelled goat anti rabbit igg h l

Mouse cardiac tissues and HUVECs were fixed with 4% paraformaldehyde. HUVECs were treated with 0.5% Triton X‐100 while cardiac tissues processed as described above, then incubated with primary anti‐α‐SMA antibody (1:200) and anti‐CD31 at 4°C after blocking with 5% BSA. The following day, cells were incubated with corresponding fluorescent antibodies: Alexa Fluor 488‐labelled Goat Anti‐Rabbit IgG (H + L) (A0423; Beyotime) and Cy3‐conjugated Goat Anti‐Mouse IgG (BA1031; Boster Biological Technology Co Ltd). Finally, nuclei were stained with DAPI. Confocal microscope was used to visualize cells.

Eyes obtained on day P17 were placed into 4% paraformaldehyde for 2 h. Then the retinas were sheared into flat mounts and blocked with 5% goat serum and 0.4% Triton X-100 for 1 h, followed by incubation with primary antibodies at 4 °C overnight. Then the retinas were washed carefully and incubated with secondary antibody combinations for 1 h. Images were taken by confocal microscopy (Zeiss, Germany). CellSens Dimension software was used to determine the number of microglia (Iba1⁺). Relative fluorescence intensity was measured by ImageJ. The following primary antibodies were used: CD31 (Abcam, ab9498, diluted 1:1000); IBA1 (WAKO, 019–19,741, diluted 1:1000); Pan-Kla (PTM-1401, diluted 1:200);YY1-K183la (PTM, diluted 1:200); YY1 (Proteintech, 66281–1-lg, diluted 1:200); p300 (Santa Cruz, sc48343, diluted 1:200); and Ki67 (Abcam, ab16667, diluted 1:200). The following secondary antibodies were used: Alexa Fluor 488-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0423); Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) (Beyotime, A0428); Cy3-labelled donkey anti-goat IgG (H + L) (Beyotime, A0502); Cy3-labelled goat anti-mouse IgG (H + L) (Beyotime, A0521); Cy3-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0516); Alexa Fluor 647-labeled goat anti-mouse IgG (H + L) (Beyotime, A0473). All secondary antibodies were diluted 1:500.