Anti gfap

Brains were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, rinsed in PBS the following day, and were subsequently placed in 30% sucrose in PBS overnight. The cryopreserved brains were then placed in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Torrance, CA) and frozen on dry ice, after which they were sectioned into 12 µm coronal slices. The following primary antibodies were used: rabbit polyclonal anti-IbaI (1:500; Wako Laboratory Chemical, Osaka, Japan), anti-GFAP (1:500; Agilent Technologies, Santa Clara, CA), anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Danvers, MA). Immunohistochemistry was performed as described previously^{91 (link)}. For biocytin detection, sectioned tissue was stained using the ABC-HRP kit (Vector Labs, Burlingame, CA) and TSA Fluorescein (PerkinElmer, Waltham, MA). Sections were nuclear counter-stained with DAPI (Molecular Probes, Eugene, OR).

Antibodies for immunofluorescence (IF) and immunohistochemistry (IHC) were obtained and used as described in the following paragraph. Anti-GFAP (Z0334, rabbit, 1:1,000 for IHC) from DAKO, Agilent Technologies (Carpinteria, CA), anti-CD31 (77699, rabbit, 1:100 for IHC) from CST, Cell Signaling Technology, anti-Ki67 (ab15580, rabbit, 1:200 for IHC) from Abcam, anti-Iba1 (019-19741 rabbit, 1: 200 for IHC and 1:250 for IF) from Wako Chemicals United States, anti-Olig2 (EMD rabbit, 1:200 for IHC) from EMD Millipore. Anti-CD8 (ab209775, rabbit, 1:200 for IF) from Abcam, anti-GrB (AF 1865, goat, 1:100 for IF) from R&D Systems, anti-Tmem119 (ab209064, rabbit, 1:200 for IF) from Abcam, and anti-F4/80 (30325T, rabbit, 1:400 for IF) from Cell Signaling Technology.

Cells were seeded on chamber slides (Ibidi) coated with poly-l-lysine and laminin (see methods for Schwann cell isolation) for 24–48 h, then fixed in 4% paraformaldehyde (EM Sciences) for 10 min at RT, permeabilized in 0.1% bovine serum albumin + 0.2% Triton X-100 for 30 min at RT, then blocked in PBS + 5% goat serum + 5% glycerol + 1% fish gelatin (all from Sigma) for 1 h at RT. Cells were incubated overnight at 4 °C in blocking buffer + rabbit anti-GFAP (Agilent Z0311), washed 3 × 5 min with PBS, then stained with Phalloidin AF594 (Thermo A12381) and anti-rabbit IgG AF488 (Thermo A11008) for 1 h at RT in the dark. Slides were then washed as above and mounted using Prolong Gold with DAPI (Thermo). DAPI, AlexaFluor488, and AlexaFluor594 were imaged with a quad excitation filter (395/476/544/640), dichroic LP beamsplitters (380/505/570), and emission filters (420LP/510-530/590-650). Images were acquired on a Nikon TiE Inverted microscope equipped with an Andor Zyla 5.5MP monochrome CMOS camera using a 20× objective and Nikon Elements acquisition software. Image resolution is 2560 × 2160 pixels (216 nm/px). Quantification of nuclei was performed using Fiji software^{79 (link)}.

Anti‐cGAS (Cell Signalling, 15102, 1:1000); anti‐STING (Cell Signalling, 13647, 1:1000); anti‐pSTING (Cell Signalling, 40818, 1:200); anti‐Tubulin (Sigma‐Aldrich, T5168, 1:2000); anti‐p21 (Cell Signalling, 2946S, 1:400); anti‐Ki67 (ThermoFisher Scientific, PA1‐21520, 1:100); anti‐NeuN (Millipore, ABN78, 1:1000); anti‐NeuN (Millipore, MAB377, 1:1000); anti‐GFAP (Sigma‐Aldrich, G3893, 1:1000); anti‐GFAP (Agilent, Z0334, 1:2000); anti‐pNF‐κB (Cell Signalling, 3033, 1:500); anti‐pIRF3 (Cell Signalling, 37829, 1:400); anti‐JNK (R&D Systems, AF1387SP, 1:1000); anti‐NG2 (Sigma‐Aldrich, AB5320, 1:200); anti‐Mouse IgG (Jackson ImmunoResearch, 715‐165‐151, 1:500); anti‐rat IgG (Jackson ImmunoResearch, 712‐605‐153, 1:500); anti‐rabbit IgG (Jackson ImmunoResearch, 711‐165‐152, 1:500); HRP anti‐Mouse IgG (Cell Signalling, 7076S, 1:10000); HRP anti‐rabbit IgG (Cell Signalling, 7074S, 1:10,000).

Immunohistochemical analyses of rat brains were performed on 5.0 μm coronal paraffin-embedded sections. Anti-GFAP (1:1000; Agilent Technologies, Santa Clara, CA, USA) and anti-ChAT (1:200; Millipore) pAbs were used to detect astrocytes and cholinergic neurons, respectively. ChAT-positive cells in the NBM were counted under a 10× objective. Five sections per animal, anteroposterior standardized with respect to the injection site and spaced 50–100 μm from one another, were analyzed. The total number of ChAT-positive cells in the QA-injected NBM was averaged, expressed as a percentage of that counted in the saline-injected NBM (n = 3 per group), and analyzed using Prism 5.0 (GraphPad Software, San Diego, CA, USA).