Carver et al.,Aged Brain Spatial Profiling - Immunofluorescence Imaging v1

This protocol provides the preparation and staining process to perform traditional immunohistochemistry on free-floating mouse brain section used in in Carver et al., "Senescent- and disease-associated microglia are modifiable features of aged brain white matter". The immunostaining process involves unconjugated- or fluorophore-conjugated- primary antibodies and secondary antibodies conjugated to fluorophores. Due to old brain accumulation of lipofuscin granules that autofluoresce, we note that in order to achieve maximal signal, a quenching step is performed to extinguish the autofluorescent noise. Sections were cut at 30 μm with a Leica CM3050 S cryostat Brain slices were incubated in free-floating solutions in 12-well or 24-well plates Imaging was performed with a Nikon Ti2 Eclipse inverted microscope and Orca Fusion BT sCMOS camera.