Rabbit anti gfap

Following treatment, the mice were anaesthetised and transcardially perfused with ice-cold PBS and fixed by the perfusion with 4% paraformaldehyde in PBS. Brain tissues were harvested and the harvested tissues were fixed in 4% paraformaldehyde again for 24 h followed by incubation in 80% ethanol. A coronal section (1 mm thick) was cut using a mouse brain slicer (Muromachi Kikai, Tokyo, Japan). Each section was embedded in paraffin. For immunofluorescence staining, 1-3 µm thick sections were de-paraffinized with xylene and rehydrated with 80% ethanol. Staining was performed using goat anti-Iba1 (Wako, Osaka, Japan), Alexa Fluor 488 conjugated rabbit anti-CD68 (Bioss Antibodies, Woburn, MA), rabbit anti-TMEM119 (Cell Signaling Technology, Danvers, MA), rat anti-BrdU (Abcam, Eugene, OR), rabbit anti-NeuN (Merck Millipore, Darmstadt, Germany), rabbit anti-GFAP (Bioss Antibodies), and rabbit anti-cleaved caspase3 (Cell Signaling Technology) antibodies. Next, the sections were incubated with Alexa Fluor 594 conjugated anti-goat IgG (Invitrogen, Waltham, MA), Alexa Fluor 594 conjugated anti-rabbit IgG (Invitrogen), or Alexa Fluor 488 conjugated anti-rat IgG (Invitrogen). Computer-assisted morphometric analysis was performed using a digital microscope controller (BZ-8000, Keyence Co., Osaka, Japan) and ImageJ software (NIH, Bethesda, MD, USA) was used for image analysis.