Fig. 4.

Characterization of primary meningeal fibroblasts by flow cytometry and immunofluorescence. a Flow cytometric analysis of primary meningeal cultures double-immunolabeled with anti-vimentin and anti-GFAP antisera. Gating (P1) of cell population is carried out from side scattering (SSC) vs forward scattering (FSC) plot of all recorder events. Majority of the gated cells (92.4%) in cultures were vimentin-positive and GFAP-negative, as seen in the lower right (LR) quadrant of the gated plot (P1). Histograms (lower panels) depict higher mean intensity of vimentin expression compared to that of GFAP. b Photomicrographs of primary meningeal culture cells double-immunolabeled for anti-vimentin (red) and anti-GFAP (green) antibodies. Cells were counterstained with DAPI (blue) to visualize nuclear staining. Arrows indicate vimentin-positive meningeal fibroblasts. c Histogram represents quantitation of the double-immunolabeled cells showing around 74.83% cells are positive for vimentin only, whereas 11.7% cells are positive for both markers and about 4.6% are GFAP only. The values are mean ± SEM from three independent experiments (having 30 different fields each) for the mentioned types of cells (one-way ANOVA; ****p < 0.0001). d Representative immunoblot showing higher level of vimentin expression in primary meningeal culture compared to mixed glial culture. All blots were re-probed with γ-actin to monitor equal protein loading