The Journal of Neuroscience, October 25, 2006, ():
Alexander Disease-Associated Glial Fibrillary Acidic Protein Mutations in Mice Induce Rosenthal Fiber Formation and a White Matter Stress Response
J. Neurosci. Hagemann et al.
26: 11162
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Supplemental Figure 1. Myelination appears normal in R236H/+ mutants. Immunostaining for myelin basic protein (MBP) in R236H/+ mice at 3 mos of age shows no overt differences in brains from mutant (B, D) and wild type (A, C) animals. Animals were anesthetized and perfused with 10 mls of PBS, followed by 50 mls of 4% paraformaldehyde. Brains were removed and post-fixed for 4hrs, cryoprotected in 30% sucrose and sectioned with a sliding microtome (40μm). Tissues were preincubated with 0.15% H2O2 in PBS with 0.25% Triton-X-100 for 10 min followed by 1% BSA in PBS with 0.25% Triton-X-100 for 2 hrs to block endogenous peroxidase activity and nonspecific binding. Sections were then incubated with goat anti-MBP (Santa Cruz #13914, diluted 1:200) overnight at 4oC, rinsed, and subsequently incubated with biotinylated rabbit anti-goat (Vector Laboratories #BA-5000, diluted 1:200) for 2 hrs at room temperature, avidin/biotin-peroxidase complex (ABC, Vector Laboratories) for 30 min., Vector SG peroxidase substrate for 5 min., and mounted on slides. Tissues were rinsed 3-4 times with PBS between incubations. Sagittal sections (A, B; 4x objective), and cerebellum (C, D; 20x objective) are shown. hi = hippocampus, cc = corpus callosum, fi = fimbria, ac = anterior commissure, mol = cerebellar molecular layer, gr = cerebellar granular layer.
- supplemental material
-
Supplemental Figure 2. Brain structure appears normal in R236H/+;GFAPTG mice. H&E histological staining of wild type (A, E), R236H/+ (B, F), GFAPTG (C, G) and double R236H/+;GFAPTG (D, H) animals shows normal anatomical features in the mutant mice at P21-22. Mice were anesthetized and perfused with 20 mls of PBS, and the brains removed for immersion fixation in methacarn overnight. Tissues were then dehydrated through graded alcohols before embedding in paraffin for sectioning and H&E staining. A-D are sagittal sections showing a cross section of the hippocampus, corpus callosum, lateral ventricle, and cortex (4x objective), whereas E-H show a higher resolution image of the dentate gyrus in the hippocampus (20x objective). Rosenthal fibers are apparent as eosinophilic aggregates in the mutant animals, especially in H. hi = hippocampus, cc = corpus callosum.
- supplemental material
-
Table 1
