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).