Protocol for autofluorescence removal in microglia by photobleaching in free-floating immunofluorescent staining of mouse brain tissue.

Autofluorescence in brain tissue poses a challenge in immunofluorescent staining by obscuring antibody-labeled proteins, mainly within microglia. Here, we describe a cost-effective protocol for removing autofluorescence in mouse brain sections by photobleaching. We outline steps to collect and section brain tissue, apply a photobleaching step using a light-emitting diode (LED), and perform immunofluorescent staining with primary and secondary antibodies. This protocol enables clearer visualization of microglial markers, particularly in mouse models of neurodegenerative diseases and aging.