For hematoxylin and eosin (H&E) staining, tissue was embedded in paraffin, cut into 5–8-μm-thick serial sections with a microtome, baked for 1 h at 65 °C in an oven and stained with H&E. For immunohistochemical staining in Fig. 1, deparaffinized and hydrated sections were boiled for 10 min in 10 mM citric acid (pH 6.0), blocked for 30 min at room temperature (RT) in 0.3% H2O2 in methanol, rinsed in dH2O, and blocked for 30 min at RT in 20% horse serum (Life Technologies Ltd, Paisley, UK) in PBS. Then, sections were incubated for 30 min at RT in either goat anti-synemin (1:200; Reglia AB, Gothenburg, Sweden) or mouse anti-GFAP (1:500, G3893; Sigma-Aldrich, St. Louis, MO, USA) in PBS plus 0.05% Tween 20 (P1379; Sigma-Aldrich), washed in PBS, incubated for 10 min at RT in biotinylated pan-specific IgG (one drop per ml PBS, BA-1300; Vector Laboratories, Burlingame, CA, USA), washed in PBS, and incubated for 30 min at RT in Vectastain ABC kit (PK-6100; Vector Laboratories). For antigen detection, sections were incubated for 8 min at RT in 3,3′-diaminobenzidine (DAB) with nickel (SK-4100; Vector Laboratories), rinsed in dH2O, incubated for 3 min at RT in eosin (1:2) in dH2O, washed in dH2O, and finally dehydrated and mounted with Vecta Mount medium (H-5000; Vector Laboratories). For combined immunohistochemical analysis (Fig. 2), sections were incubated with rabbit anti-synemin antibodies (1:200; 7) in 1% BSA in PBS; overnight at 4 °C, washed and incubated with donkey anti-rabbit Alexa 488 (1:1000, A11034; Life Technologies; 1 h at RT), washed, incubated with rabbit anti-GFAP antibodies (1:200, Z0334; DAKO, Glostrup, Denmark; overnight at 4 °C) in 1% BSA in PBS, washed, incubated for 1 h at RT in donkey anti-rabbit Alexa 555 (1:1000, A21429; Life Technologies) and TOPRO-3 (1:1000, T3604; Life Technologies) in PBS and mounted. In negative controls, the primary antibody was omitted.
Figure 1. Rosenthal fibers and reactive astrocytes in two patients with Alexander disease were positive for both glial fibrillary acidic protein (GFAP) and synemin. (A and G) Rosenthal fibers in the cortical white matter (arrows) were visualized by hematoxylin and eosin (H&E) staining. (B and H) GFAP immunohistochemical analysis in the white matter tissue showed GFAP-positive reactive astrocytes (arrows) and GFAP-positive Rosenthal fibers (C and I). Reactive astrocytes were positive for synemin (D and J arrows). Immunohistochemical analysis with antibodies against synemin combined with eosin staining showed synemin positivity in Rosenthal fibers (E and K) and in reactive astrocytes (F and L). Scale bar, 50 μm in A, B, D, G, H, J and 20 μm in C, E, F, I, K, L.
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