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. Author manuscript; available in PMC: 2014 Jul 15.
Published in final edited form as: Glia. 2013 Nov 6;62(1):78–95. doi: 10.1002/glia.22588

Fig. 2. Classical signs of reactive astrogliosis are evident in acute hippocampal slices.

Fig. 2

Comparisons of immunolabeling of GFAP, nestin, Cx43, and AQP4 in acute hippocampal slices immersion-fixed in 4% paraformaldehyde at 0 or 90 min after vibratome cutting with brains from littermates that were perfusion-fixed. All brains were harvested from 14–17 day old mouse pups. (A) GFAP immunolabeling exhibited a modest increase in slices immersion-fixed at 90 min compared with perfusion-fixed slices. Scale bar, 100 µm. Blue is DAPI stained nuclei. Insert: High magnification view of single astrocyte. (B) A histogram comparing GFAP immuno-fluorescence intensities of perfusion-fixed slices (P) with slices that were immersion-fixed immediately after vibratome cutting (0), and 90 min after cutting (90) (N=20, Mean ± sem, **, P < 0.01, ANOVA with Tukey-Kramer test). (C) GFAP intensity profile from surface to surface across the 400 µm-thick perfusion-fixed slices (black) and immersion-fixed slices (red). The intensity values of each position were normalized to overall intensity of the perfusion-fixed slice (N=10, Mean ± sem, *, P < 0.05, ANOVA with Tukey-Kramer test). (D-F) Nestin was only expressed in endothelial cells in perfusion-fixed slices, whereas essentially all astrocytes in slices immersion-fixed 90 min after preparation were nestin-positive (N=20, Mean ± sem, **, P < 0.01, ANOVA with Tukey-Kramer test). The slices were co-labeled with GFAP (green) and DAPI (blue), also shown in Merge image of one astrocyte. (G–I) The predominant astrocytic gap junction protein, Cx43, was distributed in small evenly distributed plaques in perfusion-fixed brain. A striking increase in number and size of Cx43-positive plaques was evident in acute vibratome sections fixed at 90 min. Quantification of the overall Cx43 immunofluoresence showed a significant increase in slices fixed at 90 min, with a trend toward the highest expression just below the surface of the slices (N=20, Mean ± sem, **, P < 0.01, ANOVA with Tukey-Kramer test). The slices were co-labeled with GFAP (green) and DAPI (blue), also shown in Merge image. (J–L) The astrocytic water channel, AQP4, is typically localized in astrocytic vascular endfeet in intact tissue. In vibratome sections, AQP4 redistributed and outlined the cell body and major astrocytic processes giving rise to a less polarized vascular distribution (N=20, Mean ± sem, **, P < 0.01, ANOVA with Tukey-Kramer test). The slices were co-labeled with GFAP (green) and DAPI (blue), also shown in Merge image of a single astrocyte.