Immunohistochemistry for Glyoxal/PFA-Fixed Brain

Immunohistochemistry was performed as described in our previous publications.^{56 (link)} Briefly, brain sections were fixed in cold 3% glyoxal for 30 min or 4% PFA for 20 min. After extensive washes with PBS, the sections were incubated in blocking buffer (1% BSA in PBS containing 0.3% normal donkey serum and 0.3% Triton X-100) for 1 h at room temperature. Next, the sections were incubated with primary antibodies overnight at 4°C. After extensive washes in PBS, the sections were incubated with appropriate secondary antibodies for 1 h at room temperature. Then, the sections were washed in PBS for 3 times and mounted with Fluoromount-G with DAPI. Nikon Eclipse TiE microscope and LSM710 confocal microscope were used to take images, which were further processed by ImageJ and/or Adobe Photoshop. For ICH brains, images were taken from the peri-hematoma regions.