Dapi conjugated antifade mounting medium

Frozen sections of the brain were used to determine the localization of IL-3 in the right and left cortex and hippocampus of HI rats at 24 hours by double immunofluorescence staining. The detailed procedures of immunofluorescence staining were described in our previously published study (Zhang et al., 2021). The sections were incubated with 5% goat serum (Solarbio) with 0.3% Triton X-100 (Sigma) for 2 hours at 25°C, then IL-3 (mouse, 1:200, GeneTex, SC, USA, Cat# GTX84295, RRID: AB_10728873) and neuron-specific nuclear protein (NeuN) (rabbit, 1:200, GeneTex, Cat# GTX132974, RRID: AB_2886793) or glial fibrillary acidic protein (GFAP) (rabbit, 1:500, GeneTex, Cat# GTX108711, RRID: AB_2037091) for 18 hours at 4°C. Then, secondary antibodies Dylight 594 (goat anti-mouse IgG, 1:200, Abbkine, Cat# A23410) and Dylight 488 (goat anti-rabbit IgG, 1:200, Abbkine, Cat# A23220, RRID: AB 2737289) were added for incubation at 25°C for 3 hours. The sections were sealed with DAPI-conjugated Antifade Mounting Medium (Beyotime) and observed at 200× under a fluorescence microscope.

HpcMSCs were resuspended in 0.25% trypsin (Gibco) and then seeded into a 96-well plate at a density of 4000 cells per well. After 48 hours of culturing, the cells were washed twice with 0.01 mM PBS and fixed with 4% paraformaldehyde (Biosharp) for 20 minutes. Subsequently, cells were washed three times with 0.01 mM PBS for 5 minutes each time, and then 100 μL of 5% goat serum (Solarbio, Beijing, China) with 0.3% Triton-X100 (Sigma, Shanghai, China) was added to each well at 37°C for 30 minutes. The primary antibodies CD45 (mouse, 1:100, Bioss, Beijing, China, Cat# bsm-33052M, RRID: AB_2939047) and CD90 (rabbit, 1:100, Bioss, Cat# bs-0778R, RRID: AB_10857837) or CD44 (rabbit, 1:100, Bioss, Cat# bs-2783R, RRID: AB_10854140) were incubated for 18 hours at 4°C. Afterwards, secondary antibodies Dylight 594 (goat anti-mouse IgG, 1:200, Abbkine, Wuhan, Hubei Province, China, Cat# A23410) and Dylight 488 (goat anti-rabbit IgG, 1:200, Abbkine, Cat# A23220, RRID: AB 2737289) were added for incubation at 37°C for 1 hour. The cells were sealed with 4′,6-diamidino-2-phenylindole (DAPI)-conjugated Antifade Mounting Medium (Beyotime, Shanghai, China) and observed at 400× under a fluorescence microscope (Leica, Wizzler, Hesse, Germany).