Ab150062

In brief, rats were anaesthetized and transcardially perfused with saline and 4% paraformaldehyde. Brains were wholly removed to prepare frozen coronal sections of 10 μm in thickness. For Nissl's staining, slices were incubated with Nissl's staining solution (Beyotime) for 30 min at 37 °C and gently washed with PBS 7.4 for scanning microscope imaging (Olympus). For immunofluorescence staining, slices were incubated with primary antibodies (anti-Iba1 antibody, 1:200, ab5076, Abcam; anti-GFAP antibody, 1:4000, ab7260, Abcam) and then secondary antibodies (donkey anti-goat IgG Alexa Flour 488, ab150129, Abcam; donkey anti-rabbit IgG Alexa Flour 555, ab150062, Abcam) for scanning fluorescence microscope imaging (Olympus). Meanwhile, DAPI (Beyotime) was applied to direct nuclei.

To verify the proper placement of the optical fibers (fig. S21), animals are deeply anesthetized and transcardially perfused with 0.1 M PBS, followed by 4% paraformaldehyde-borate fixative (pH 7.4) for 20 min. Brains are removed and then postfixed in the same fixative overnight at 4°C, followed by cryoprotection in 10, 20, and 30% sucrose solution at 4°C before being processed for immunocytochemistry. Brains are sliced into 35-μm sections with a cryostat microtome (CM3050 S, Leica). The sections are collected into four serially ordered sets of sections in PBS. For histology examination, the brain sections are washed in PBS and coverslipped with VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (F6057, Sigma-Aldrich). To verify the inflammatory response (fig. S13), after an initial blocking step (3% normal donkey serum in 0.01 M PBS containing 1% Triton X-100), the sections are incubated with the following primary antibodies for 24 hours at 4°C: rabbit anti-NeuN (1:1000; ab177487, Abcam) and mouse anti-SOX9 (1:1000; ab76997, Abcam). The sections are washed and incubated with Alexa Fluor 647 donkey anti-mouse immunoglobulin G (IgG; 1:1000, ab150107, Abcam) and Alexa Fluor 555 donkey anti-rabbit IgG (1:1000, ab150062, Abcam) for 2 hours at approximately 25°C. The fluorescence images are captured using a confocal microscope (FV3000, Olympus).