Chicken anti gfap

Cultures were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Cultures were incubated with phosphate buffered saline (PBS) containing 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at RT and then with blocking solution (PBS containing 2% bovine serum albumin (Sigma-Aldrich)) for 30 min at RT with gentle shaking. Subsequently, cultures were incubated for 2–3 hrs at RT with mouse anti-β3 tubulin (TuJ1 clone, 1:2000, R&D Systems, Inc., Minneapolis, USA) and chicken anti-GFAP (1:5000, Millipore) antibodies. After washing (3 × 10 min in PBS at RT) cultures were incubated for 2 hrs at RT with 488 and 549 DyLight dyes conjugated to affinity-purified secondary antibodies (1:1000, Jackson ImmunoResearch). Hoechst 33258 (0.4 μg/ml, Sigma-Aldrich) was used for nuclear counterstaining.

The following primary antibodies were used: rabbit anti-cFos (diluted 1:2000; Cell Signaling Technologies, Cat# 2250, MA, USA), rat anti-FLAG (diluted 1:500; BioLegend, Cat# 637303, CA, USA), chicken anti-GFAP (diluted 1:1000; Millipore, Cat# AB5541), chicken anti-GFP (diluted 1:1000; Invitrogen, Cat# A10262).

Cultures were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Cultures were incubated with phosphate buffered saline (PBS) containing 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at RT and then with blocking solution (PBS containing 2% bovine serum albumin (Sigma-Aldrich)) for 30 min at RT with gentle shaking. Subsequently, cultures were incubated for 2–3 hours a RT with mouse anti-β3 tubulin (TuJ1 clone, 1:2000, R&D Systems, Inc., Minneapolis, USA) and chicken anti-GFAP (1:5000, Millipore) antibodies. After washing (3 × 10 min in PBS at RT) cultures were incubated for 2-hours at RT with 488 and 549 DyLight dyes conjugated to affinity-purified secondary antibodies (1:1000, Jackson ImmunoResearch). Hoechst 33258 (0.4 μg/ml, Sigma-Aldrich) was used for nuclear counterstaining. For F-actin staining, cultures were incubated for 2–3 hours a RT with 488 Fluorescent Phalloidin (1:500 in PBS; Cytoskeleton, Inc).

Frozen sections (20 μm) were first incubated with goat anti-mouse serpinA3N (R&D Systems cat. no. AF4709) at a dilution of 1:1000 overnight at 4 °C then incubated for 30 min at room temperature with biotinylated anti-goat IgG (Vector Labs cat. no. PK-6105) according to the manufacturer’s instructions followed by ABC reagent (Vector Labs cat. no. PK-6105) and stained with 3,3′-diaminobenzidine (DAB) substrate solution (brown) (Vector Labs cat. no. SK-4100). Sections were then thoroughly washed and incubated with either chicken anti-GFAP (Millipore, UK cat. no. AB5541), rabbit anti-IbA1 (Wako cat. no. 019-19741), rabbit anti-NPY (ThermoFisher Scientific cat. no. PA5-19568) or rabbit anti-AgRP (Phoenix Pharmaceuticals cat. no. H-003-57) at a dilution of 1:300 overnight at 4 °C and then at room temperature for 30 min with either goat anti-chicken IgG (Vector Labs cat. no. BA-9010) at a dilution of 1:200 followed by Vectastain Elite ABC kit (goat IgG) (Vector Labs cat. no. PK-6105) or directly with the Vectastain Elite ABC kit (goat IgG) and stained with Vector SG (blue) (Vector Labs cat. no. SK-4700). Control sections omitted the primary antibodies.

Seven days after CCI, the mice were sacrificed, and their spinal cords were removed and postfixed in 4% paraformaldehyde (PFA) overnight at 4°C. After dehydration, the tissues were paraffin embedded and sectioned (7 μM) on a microtome (Leica, RM45). Adjacent coronal sections from corresponding regions of the lumbar (L4 to L6) spinal cords of naive and CCI mice were incubated overnight at 4°C with the following primary antibodies: rabbit anti-XCL1 (1:50, Novus Biologicals), rabbit anti-ITGA9 (1:50, Abcam), rabbit anti-XCR1 (1:50, Lifespan Biosciences), mouse anti-NeuN (1:250, Merck), rat anti-IBA1 (1:1000, Abcam), and chicken anti-GFAP (1:10000, Merck). Antigen-bound primary antibodies were visualized with appropriate Alexa Fluor 488/594–conjugated donkey secondary antibodies (1:100, Invitrogen). Hoechst 33342 (Invitrogen) was used to stain cell nuclei. Stained sections were examined and acquired under a high-class confocal microscope (Leica TCS SP8 WLL) equipped with HyD, PMT and TLD detectors. The ipsilateral part of the lumbar spinal cord was visualized on representative images.