Rabbit polyclonal anti cathepsin b

Three hours post-reperfusion, the rats were deeply anesthetized before sequential perfusion with ice-cold saline and 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed and dehydrated in a sucrose gradient (20%–30%) in PBS at 4 °C. Coronal sections (10 μm thick) at bregma ± 2 mm were cut on a cryostat and stored at −20 °C until further use. The slices were blocked with 3% normal goat serum in 0.5% Triton X-100 for 30 min at room temperature. Subsequently, the slices were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 2 h at room temperature. The following antibodies were used: rabbit polyclonal anti-cathepsin B and mouse monoclonal anti-NeuN (both 1:1000, Abcam). DAPI (4’,6-diamidino-2-phenylindole; 1:1000; Sigma-Aldrich) was used to stain nuclei. The following secondary antibodies were used: green-fluorescent Alexa Fluor 488-conjugated donkey anti-mouse and red-fluorescent Alexa Fluor 594-conjugated donkey anti-rabbit (both 1:1000; Abcam). Fluorescent signals in the right penumbra region of cortex (2 mm lateral from the midline) were detected using confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan).