Fig. 6: Engulfment of endogenous synaptic proteins in the optic nerve crush paradigm by cells harvested from perfusion-fixed tissue.

a Experimental schematic depicting isolation of cells from perfusion-fixed tissue and assessment of engulfment following optic nerve crush (ONC). Prior to bilateral ONC, retinal ganglion cells in both eyes were labeled by intravitreal injection of cholera toxin subunit-B conjugated with AF488 (CTB-AF488) to facilitate the micro-dissection of the lateral geniculate nuclei and superior colliculi (LGNs/SCs). AF488 and SNAP-25 signals were compared between no crush and ONC for both microglia (b–c) and BAMs (d–e). Flow cytometry plots display AF488 and SNAP-25 signals in microglia and BAMs. The gates for AF488⁺ cells were based on the fluorescent signal from WT cortical cells while the gates for SNAP-25⁺ cells were based on the fluorescence of their respective isotype controls (<1% cells in positive gates). Cells were gated on DAPI (to identify nucleated cells), single cells, CD45⁺, CD68⁺, and GR1^-. Microglia were further gated on CX3CR1^high and P2Y12^high, while BAMS were gated on CD206^high and CD38^high. The MFI for AF488 and SNAP-25 was normalized to the mean MFI of no crush controls and depicted for all microglia (b–c) and BAMs (d–e) independent of the gates shown on the plots. n = 5 mice (3 females and 2 males) per condition. Error bars depict standard error of the mean. Statistical analysis: unpaired two-tailed t test. Source data provided.