Immunofluorescence Staining of HIF-1α and RECA-1 in Cerebral Ischemia

The rats were immediately perfused with PBS and 4% PFA after cerebral ischemia for 2 hours. We followed the previously published methods of Shen et al. in doing immunofluorescence staining [13 (link)]. In brief, 20 μm thick cryosections were preincubated for 1 hour at room temperature in PBS which contained 0.1% Triton X-100, 1% BSA, and 5% goat serum (Solarbio, Beijing, China) to cover nonspecific binding sites. The HIF-1α (1 : 200, Abcam) and RECA-1 (1 : 100, Abcam) primary antibodies were applied to the brain slices and incubated overnight at 4°C. Appropriate secondary antibodies that bind to Cy3 (anti-mouse, 1 : 800) or 488 (anti-rabbit, 1 : 800) were used for detection. The nucleus was stained with Dapi. The LSM 700 confocal laser scanning microscope (Zeiss) was used to take images from the ischemic area and the mirror nonischemic area [13 (link)].