Nissl staining
Nissl staining provided information about the pathology of hippocampal neurons.The brain tissue was fixed for 24h with 4% paraformaldehyde, ethanol dehydrated paraffin-embedded, coronal sectioned, deparaffinized by immersing it in 1% toluidine blue solution for 40min, washing three times with distilled water, ethanol dehydrated, transparent with xylene, and neutral resin sealed. An optical microscope was used to observe the Nissl body in the CA1 region of the hippocampus.
SA-β-gal staining
Because the buildup of endogenous lysosomal galactosidase is a hallmark of aging cells, we determined the senescence-related β-galactosidase (SA-β-gal), which is widely used in mammalian cells as a senescence biomarker (Zhang MS et al,2015). The Leica cryostat was used to prepare a frozen segment of the coronal hippocampus of brain tissue. The section and third-generation neurosphere were fixed for 30 minutes at room temperature with the kit fixative solution, washed 3 times with PBS, stained with the β-galactosidase staining working solution, and incubated overnight at 37°C. Using an optical microscope, the hippocampal CA1 region and NSCs were observed. The blue-stained cells were senescent cells or positive cells.