Immunofluorescence Staining for Cellular Markers

Immunofluorescence staining was performed as previously described (Chen et al., 2020 (link)). In short, the frozen sections were rewarmed at room temperature. After antigen retrieval in citric acid antigen repair solution, the sections were incubated at 4°C overnight with the following primary antibodies: anti-CD31 (1:200, ab222783, Abcam, USA), anti-IBA-1 (1:400, 019-19741, FUJIFILM Wako Shibayagi, Japan), anti-CD68 (1:200, 28058-1-AP, Proteintech, China), anti-GFAP (1:400, #80788, CST, USA), anti-MPO (1:50, A1374, Abclonal, China), and anti-NeuN (1:200, #94403, CST, USA). Then, the sections were incubated with the appropriate fluorescently labeled secondary antibody at room temperature for 1 h. For double staining with TUNEL fluorescence, the procedure followed the instructions of the CF488 TUNEL Cell Apoptosis Detection Kit (G1504, Servicebio, Wuhan, China). After incubation with secondary antibody, the sections were equilibrated with Equilibrium Buffer for 10 min and then incubated at 37°C for 1 h with TDT incubation buffer. Finally, nuclei were stained with DAPI. The sections were observed and photographed with a fluorescence microscope (U-HGLGPS, Olympus, Japan). Micrographs were analyzed with ImageJ software (ImageJ, RRID: SCR _ 003070).