2.6 Immunohistochemistry and immunofluorescence
Mice were anesthetized and transcardially perfused with ice-cold heparin saline, followed immediately by a 4% formaldehyde solution. The brain was subsequently removed and immersed in a 4% formaldehyde solution, as well as 15% and 30% sucrose solutions overnight at 4℃ respectively. Afterwards, the tissues were sectioned into coronal slices of 16μm thickness using a cryostat microtome (Leica, Wetzlar, Germany). HT22 cells were cultured on cell slides in 6-well plates and fixed in 4% paraformaldehyde for 15 minutes at room temperature. The brain sections were washed in PBS for 30 minutes, and the cells were rinsed with PBS. All samples were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes and blocked with 5% bovine serum albumin (BSA) (Solarbio, Beijing, China) for 30 minutes at room temperature before being rinsed three times in PBS. For antibody staining, sections were incubated overnight at 4 ℃ with primary antibodies against galectin-3 (1:250, abcam, USA) and Neun (1:200, Cell Signaling Technology, USA). After washing the samples in PBS for 10 minutes, they were incubated with fluorescent secondary antibodies (1:500, Invitrogen, USA) for 1 hour at 37 ℃. Finally, slices were overlaid with a mounting medium containing DAPI to counterstain nuclei. The immunofluorescent images were captured using an Olympus fluorescence microscope in Tokyo, Japan. Five random regions of each sample were imaged and analyzed using Image J software (National Institutes of Health, USA).