Fluorescent Staining of Caspase-3 in Cerebral I/R

Double fluorescent staining was performed to examine caspase-3 activity in microglial cells following cerebral I/R as described previously 9 (link),13 (link). Briefly, brain tissues were immersion-fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut at 7 μm, and stained with a specific anti-cleaved caspase-3 antibody which was labelled with FITC. After washing, the sections were incubated with anti-ionized calcium-binding adapter molecule 1 (IBA1; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 25°C for 1 hr to stain activated microglial cells. After washing, the sections were incubated with Texas Red conjugated anti-goat antibodies (sc-2783; Santa Cruz, Santa Cruz, CA, USA) for 1 hr at 25°C. The sections were then incubated with DAPI for staining the nucleus. The sections were covered with fluorescence mounting medium (Vector Labs, Burlingame, CA, USA). The images were viewed on an EVOS-fI digital inverted fluorescent microcopy (Advanced Microscopy Group, Bothell, WA, USA). Fields of cortex were randomly examined using a defined rectangular field area for analysis of microglia activation.