Mice were perfused transcardially with 0.9% sodium chloride followed by 4% paraformaldehyde after anesthetized with 2% pentobarbital sodium (Sigma-Aldrich, USA). Brain samples were collected, fixed in 10% neutral formalin, embedded in paraffin, and cut into 3 μm-thick sections. Brain sections were then de-paraffinized in xylene, rehydrated via graded alcohols and stained with hematoxylin and eosin (H&E) (Biosharp, Wuhan, China). The sections were observed and photographed under a light microscope (Leica, Heidelberg, Germany).
For immunohistochemistry (IHC) analysis, brain sections were subjected to antigen retrieval by boiling the slices in citrate buffer (pH 6.0) with high heat for 15 min. Then sections were treated with 3% H2O2 for 10 min to remove endogenous peroxidase, blocked with 5% rabbit serum at room temperature for 20 min, and incubated with rabbit anti-mouse CD49b monoclonal antibody (mAb) (Abcam, Cambridge, UK) at 4°C overnight. After being washed in PBS, the sections were incubated with an HRP-conjugated secondary antibody (DAKO, Glostrup, Denmark) at room temperature for 15 min and then stained with 3, 3’-diaminobenzidine (DAB) for 10 min. Haematoxylin was used for cell nuclei detection. The sections were visualized and digitally scanned with a light microscope.
For immunohistochemistry (IHC) analysis, brain sections were subjected to antigen retrieval by boiling the slices in citrate buffer (pH 6.0) with high heat for 15 min. Then sections were treated with 3% H2O2 for 10 min to remove endogenous peroxidase, blocked with 5% rabbit serum at room temperature for 20 min, and incubated with rabbit anti-mouse CD49b monoclonal antibody (mAb) (Abcam, Cambridge, UK) at 4°C overnight. After being washed in PBS, the sections were incubated with an HRP-conjugated secondary antibody (DAKO, Glostrup, Denmark) at room temperature for 15 min and then stained with 3, 3’-diaminobenzidine (DAB) for 10 min. Haematoxylin was used for cell nuclei detection. The sections were visualized and digitally scanned with a light microscope.