Planc n

Whole brain imaging for GFAP slides [Ta (n = 6) At (n = 4)] was scanned with a Ventana-DP 200 brightfield tissue scanner microscope (Roche), equipped with a 40x objective (NA 0.75; UPlanSApo; Olympus) using a 40x digital zoom at a single layer.
Unique cell imaging for fractal morphological analysis of astrocytes was performed using brightfield microscope imaging. For this purpose, five cells were recorded per telencephalic region in the pallium (Laminar edge of pallium (LEP) and vascular pallium), the pallidum, and the mesencephalon for Ta (n = 6) and At (n = 3). These cells were randomly selected and captured using an Olympus CX35 microscope (Model X31RBSFA) equipped with a 100x oil immersion objective (NA 1.25; PlanC N; Olympus) and a Swift camera (SC1803R).

Immunohistochemistry/immunofluorescence was performed as follows: cryosections were incubated with blocking buffer (5% goat serum, 5% donkey serum, 0.5% BSA, 0.25% Triton-X 100 in PBS) for 1 h and labeled with primary antibodies (Supplementary Table S2) or isotype controls overnight at 4°C in blocking buffer. The following day, sections were washed three times with washing buffer (0.2% Tween-20 in PBS) and incubated with fluorescence probe-conjugated secondary antibodies for 1 h at room temperature. We used mannose receptor-1 (CD206) as a marker for resident macrophages and platelet-derived growth factor receptor α (PDGFRα) as a marker for FAPs. The TSA Green kit (Tyramide Signal Amplification; Perkin Elmer, Waltham, MA) was used for CD206 and PFGFRα staining after 1 h of incubation with biotinylated donkey-anti-rabbit F(ab′)2 IgG fragments (2.5 µg/ml) to enhance the immunostaining signal. Nuclei were then stained with DAPI (1 µg/ml) and mounted using Vectashield (Vector Labs, Burlingame, CA). All images were taken on a Revolve Echo widefield fluorescence microscope using a x10 (PlanC N, Olympus) or a x20 objective (UPlanFL N, Olympus) and 5 MP CMOS Monochrome Camera.