Anti gfap antibody

Astrocyte proliferation was determined using the Cell-Light EdU (5-ethynyl-2′-deoxyuridine) Apollo567 In Vitro Kit (#C10310; Riobio, Guangzhou, China) following the manufacturer’s instructions. SB216763 or Nec-1 were added to the medium during reoxygenation and the astrocytes were incubated for 24 h. Cells were sequentially incubated with 20 μM EdU during reoxygenation; 4% paraformaldehyde (to fix the cells for morphological analysis) for 30 min at room temperature; and 0.5% Triton X-100 (to permeabilize the cells) for 10 min. Finally, 1× Apollo reaction solution was added and the cells were incubated for 30 min. To label GFAP, cells were exposed overnight to an anti-GFAP antibody (1:1,000, #53-9892-82, Thermo, MA, USA) at 4°C. Hoechst (1:5,000; #33258; Sigma-Aldrich, MO, USA) was used to stain the nuclei. Images were obtained using a confocal microscope (LSM 710; Carl Zeiss, Jena, Germany).

Brain sections of 4 μm were collected on Superfrost slides for GFAP staining and dried at 37 °C for 1 day. After deparaffination and hydration, sections were autoclaved for 30 min in 0.2% of citrate buffer solution at pH 6.2 at 121 °C. Next, endogenous peroxidases were blocked with a 3% hydrogen peroxide solution for 20 min, while non-specific antibody binding sites were blocked using 6% normal goat serum for 1 h. Immunohistochemical detection of GFAP was performed by incubating sections overnight at 4 °C with an anti-GFAP antibody (Thermo-Scientific, Waltham, MA, USA, 1:200). After washing in PBS with 0.1% Tween-20 and incubating with the appropriate biotinylated secondary antibody (goat anti-rabbit Vector Labs, Burlingame, CA 1:200), sections were treated with ABC Complex (Vector) for 1 h. Lastly, sections were stained with diaminobenzidine (Dako-Cytomation, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin. Gfap-labeled sections designated for morphometric analysis were not counterstained. Each IHC run included positive and negative control sections.