Anti trpv1

Immunofluorescence staining was carried out to detect GFAP expression in mice after HIBD as well as TRPV1, GFAP, and iNOS expression in astrocytes after OGD. Brain serial coronal sections and astrocytes were washed with PBS before fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, they were incubated with a blocking solution (5% FBS) for 30 min at 37 °C. Then, they were incubated with the anti-TRPV1 (Novus Biologicals, #9886, 1:300), anti-GFAP (Cell Signaling, #3670, 1:200), or anti-iNOS (Cell Signaling, #2985, 1:200) antibodies overnight at 4 °C. On the following day, they were washed and incubated with secondary antibodies Cy3-conjugated anti-IgG (Protein tech, SA00009-4, 1:30), Alexa Fluor® 488 Conjugates (Cell Signaling Technology, #4408, 1:200) for 1 h at 37 °C and DAPI (Beyotime, #C1002,1:2000) for 1 min at room temperature. Phalloidin staining was performed following the same protocol used for immunofluorescence (IF) while astrocytes were incubated with FITC-phalloidin (sigma, #p5282, 5 μg/ml) for 1 h and DAPI for 1 min at 37 °C in the dark. Images were obtained using a confocal microscope (Leica-LCS-SP8-STED).

Astrocytic proteins were extracted, and the protein concentrations were measured. Equal amounts of protein were loaded on an SDS-PAGE gel. After electrophoresis and transfer to a polyvinylidene fluoride (PVDF) membrane, the membranes were blocked by 5% skimmed milk for 2 h. The membranes were incubated with the anti-TRPV1 (Novus biologicals, #9886, 1:1000), anti-GFAP (Cell Signaling, #3670, 1:1000), anti-GAPDH (Abcam, #9485, 1:1000), anti-ARG-1(Cell Signaling, #9819, 1:1000), and anti-β-actin (Protein tech, #60008, 1:10000) antibody overnight at 4 °C. Corresponding secondary antibodies were incubated for 1 h at room temperature. The reaction was detected in a chemiluminescent reagent (ECL; Menlo Park, California, USA).