Rabbit anti ire1α

After survival periods of 1 and 7 days, five mice from each group were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with 0.9% saline, followed by fixation with 4% paraformaldehyde. Brain samples were removed and postfixed in 4% paraformaldehyde overnight at 4°C, followed by cryoprotection with 15%, 20%, 30% gradient sucrose solution for 48 hr at 4°C, respectively. Serial coronal sections (25 μm) were prepared using a freezing microtome. Before immunofluorescence staining, sections were rinsed three times with phosphate buffered saline (PBS) for 10 min and blocked for 2 hr with 10% goat serum in PBS containing 0.1% Triton X‐100. Sections were incubated overnight at 4°C with primary antibodies: rabbit anti‐CHOP (1:200, Abcam, Cambridge, UK), rabbit anti‐IRE‐1α (1:200, Cell Signaling Technology) and mouse anti‐NEUN (1:400, Abcam, Cambridge, UK). After primary antibody incubation, sections were rinsed three times in PBS for 10 min, followed by incubation with the appropriate Alexa Fluor‐tagged secondary antibody. DAPI was used for counterstaining. Sections were imaged using a Carl Zeiss confocal laser microscopy.