Fig. 4
From: Impacts of autofluorescence on fluorescence based techniques to study microglia
Imaging of autofluorescent granules in microglia on fixed brain slices. A Diagram illustrates the imaging of hippocampus CA1 microglia on fixed brain slices. B Representative 3D reconstructed images of a microglia from 2 months old Cx3cr1GFP mice with autofluorescence (AF) imaged in TRITC channel (561 nm laser, 570–620 nm band pass filter). C Representative 3D reconstructed images of a microglia stained with CD68 from Cx3cr1GFP mice. The arrow points to a CD68+ lysosome containing an autofluorescent granule in soma (AF +); while the arrowhead points to CD68+ lysosomes located in process without autofluorescent granules (AF-). D Volume of AF+ and AF− CD68 + lysosomes as in panel (C) (n = 15 cells from 3 mice, Mann–Whitney test). E Images of an autofluorescent granule from a GFP expressing microglia through continuous emission filter excited by 566 nm laser and its corresponding emission spectrum (n = 8 cells from 3 mice). F Images of an autofluorescent granule from a microglia in wild type mouse labeled by Iba-1 antibody through continuous emission filter excited by 488 nm laser and its corresponding emission spectrum. The emission spectrum of FITC, PE, and APC were shown for comparison (n = 8 cells from 3 mice). G Z projected stack images of microglia from Cx3cr1GFP mouse with autofluorescence imaged as in panel (B). H Z projected stack images of microglia on slices with the treatment of TrueBlack® lipofuscin autofluorescence quencher before imaging. I Values of autofluorescence in microglial somata as in (G) and (H) (n = 17 cells from 3 mice, 2 tailed t-test). Bars represent means ± SD. ***P < 0.001