Figure 6

IFNβ-producing microglia act as orchestrators of myelin debris removal. a Representative immunofluorescent stainings of primary adult microglia prepared as described above. Cells were co-stained for YFP, pIRF7, and Mac3 together with DAPI. Mac3 and pIRF7 were depicted on separate images for better visualization. Scale bar represents 50 μm. b Scheme of the experimental setup. Primary microglia cells were isolated from IFNβ^mob/mob mice and treated with poly (I:C) for 24 h. Stimulated microglia (CD11b and CD45) were sorted by flow cytometry for YFP and labeled with DiD. For co-culture experiments YFP-positive and YFP-negative sorted microglia cells were transplanted onto demyelinated cerebellar slice cultures isolated from WT, IFNβ^−/− and IFNAR1^−/− mice. Demyelination was performed one day prior of transplantation with LPC. c Representative image of transplanted DiD labeled microglia in close vicinity to endogenous microglia in the OSC. Scale bar represents 50 μm. d Representative images of YFP-negative (left) and YFP-positive (right) sorted microglia after transplantation onto demyelinated OSCs. Scale bar represents 50 μm. e Diagram shows intensity of immunostaining for MBP in OSCs from WT, IFNβ^−/− and IFNAR1^−/− mice transplanted with YFP-negative (white bars) and YFP-positive (black bars) microglia. The quantification of myelin debris in areas surrounding transplanted cells in demyelinated areas was performed by histological analysis of MBP intensity in 0.01 mm² with ImageJ software (n = 4 for WT; n = 3 for IFNβ^−/− and n = 3 for IFNAR1^−/−). Data shown as mean + SEM. Statistical significance was determined by Student’s t test with *p < 0.05.