Anti gfap antibody

Astrocytes on coverslips were fixed with 4% formaldehyde for 30 min. Anti‐GFAP antibody (1:100, Cell Signaling Technology, Cat: 3670S) was incubated overnight at 4°C, and anti‐mouse Alexa Fluor® 488 (1:1000, Abcam, Cat: ab150113) was incubated for 2 h at room temperature. Images were captured by the Thunder Imaging System (Leica, Germany). The astrocytes were counted with ImageJ software version 1.53c (NIH, USA).

Microglia were isolated from brain tissue using the previously described Percoll-gradient isolation technique65 (link). Briefly, brains were removed from perfused mice, divided into ipsilateral and contralateral hemispheres, minced and digested by incubation with 250 μg ml⁻¹ collagenase IV/DNase I at 37 °C for 45 min each. The resulting cell suspensions were fractionated on 50/70% Percoll gradients at 1,000 g for 25 min. Microglial cells were collected from the interface between the 50 and 70% bands and washed with hanks' balanced salt solutions (HBSS, Welgene). The purity of the isolated microglial cells was determined by FACS analysis. Astrocytes were isolated as previously described66 . In brief, cell suspensions from brain tissues were fractionated on 30/60% Percoll gradients at 1,000 g for 25 min. Astrocytes were collected from the PBS/30% interface. The purity of the isolated astrocytes was determined by FACS analysis using an anti-GFAP antibody (Cell Signaling Technology, #3670, 1:500).

Slice cultures were left attached on the Millicell membrane and stained as free-floating sections in 6-well plates. Cultures were first fixed with 4% paraformaldehyde in PBS (PAA, Austria) for 2 h at 4°C. After washing with cold PBS, slices were permeabilized by 0.4% TritonX-100/PBS for 90 min at RT. Slices were then blocked with 5% BSA for 2 h and afterwards incubated with primary antibody diluted in PBS for 2-3 days at 4°C. After washing with PBS, slices were incubated with secondary antibody for 2 days at 4°C. After washing, slices were mounted with Permafluor mounting solution (Beckman Coulter, Paris, France), cover-slipped and dried before imaging. The following primary antibodies were used: monoclonal anti-neuronal nuclei (NeuN) antibody (Chemicon International, Temecula, CA) (1:1000), pan-Tau antibody K9JA (Dako, Hamburg, Germany, Nr. A0024 (1:1000)); anti-Iba1 antibody (Wako Chemicals, Germany) (1:1000); anti-GFAP antibody (Cell signaling technology) (1:1000), anti-BrdU (GenTex) (1:1000), DCX (Cell Signalling) (1:500) and Ki67 (Abcam) (1:500). All fluorescent (goat anti-rabbit/mouse cyanine 2, 3 and 5)-labeled secondary antibodies were from Dianova (Hamburg, Germany) (1:1000).