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.