Ix71 inverted microscope system

Double-immunofluorescence Staining was conducted as previously described (Lu et al., 2018 (link), 2019 (link)). Briefly, brain samples dehydrated with 30% sucrose at least 24 h and tissue sections (8 μm thickness) were cut with a cryostat. The slides were incubated in 5% blocking buffer for 2 h at room temperature, then incubated with rabbit anti-CD86 antibody (1:100) or anti-CD206 antibody (1:100), respectively, overnight at 4°C. Afterward, incubated with another primary antibody, namely mouse anti-Iba-1 (1:50), under similar conditions. Alexa-Fluor 594 goat-rabbit IgG and Alexa-Fluor 488 donkey-mouse IgG were used as the corresponding secondary antibodies for 1 h at the room temperature. Then the slides were stained with 4-diamidino-2-phenylindole (DAPI) for 15 min to show the location of nucleus. The fluorescently stained cells were imaged on an Olympus IX71 inverted microscope system and analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, United States).

For TUNEL staining, brain sections were incubated in 4% paraformaldehyde blocking for 30 min at room temperature, then incubated with rabbit anti-NeuN (1:200) overnight at 4°C. Afterward, Alexa-Fluor 488 goat-rabbit IgG was used as the corresponding secondary antibodies for 1 h at the room temperature. The sections were incubated with the TUNEL reagent (Beyotime Biotechnology, Shanghai, China) for 1 h at 37°C. Then the slides were stained with DAPI for 15 min to show the location of nucleus. The number of TUNEL-positive neurons was regarded as apoptosis index. The fluorescently stained cells were imaged on an Olympus IX71 inverted microscope system and analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, United States).

Immunofluorescence staining was performed as previously described^{24 (link)}. Briefly, the immunostaining protocol was as follows: rats were deeply euthanized and perfused with 4% paraformaldehyde in 0.1 mM phosphate-buffered saline (PBS, pH 7.4). Brain samples were immersed in 30% sucrose until sinking to the bottom. 8 µm-thick slices were cut with a cryostat. The slices of each coronal sections were incubated in blocking buffer for 2 h, then washed with PBS three times for 10 min. Next, the slices were incubated with anti-MFG-E8 (1:200) and anti-cleaved caspase-3 antibody (1:100) respectively, in a dark place overnight at 4 °C. Afterwards, the slices were washed three times with PBS and incubated with another antibody, namely anti-NeuN (1:100), anti-GFAP (1:100), anti-Iba-1 (1:50), under similar conditions. The following day, the slices were thoroughly washed with PBS and incubated with the corresponding secondary antibodies for 1 h. After the wash with PBS, the slices were stained with DAPI for 2 min to show the location of the nucleus. Coverslips were applied with mounting media. The fluorescently-stained cells were imaged on an Olympus IX71 inverted microscope system and analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). All of the processes were conducted by two investigators who were blinded to the grouping.