Mouse anti cd206

The immunofluorescence assay was performed with modifications, as previously described [45 (link)]. Briefly, cryosections and BMDM were washed with tris-buffered saline (TBS) and fixed in 4% paraformaldehyde (PFA) for 10 min. After washing, the samples were permeabilized with TBST (0.2% Triton X-100 in TBS) for 10 min and washed three times with TBS for 5 min. Samples were blocked using 2% BSA and incubated at 4°C overnight with primary antibodies, including rabbit anti-Nogo-A (Abcam, catalog no. ab62024), mouse anti-CD68 (Santa Cruz Biotechnology, catalog no. ab955), mouse anti-iNOS (Santa Cruz Biotechnology, ab49999), mouse anti-CD206 (Santa Cruz Biotechnology, catalog no. sc-58986), mouse anti-CHOP (Santa Cruz Biotechnology, sc-71136), and mouse anti-calnexin (Novus Biologicals, catalog no. NB300518). After three washes with TBS for 5 min, samples were incubated with secondary antibodies (donkey anti-mouse immunoglobulins (Alexa Fluor 488, Abcam, catalog no. ab150105) and donkey anti-rabbit immunoglobulins (Alexa Fluor 555, Abcam, catalog no. ab150066) for 1 h in the dark. The samples were mounted using ProLong™ Gold Antifade reagent containing DAPI to visualize the nuclei (Cell Signaling Technology, catalog no. 8961s) and were analyzed by confocal microscopy (ZEISS).

Sections were immersed in 3% hydrogen peroxide for 15 min. Nonspecific antigen blocking was performed in 2% bovine serum albumin (BSA) for 1 hour. Sections were then incubated overnight at 4 °C with rabbit anti-Ki67 (1:200, Abcam), rabbit anti-CD31 (1:200, Bioworld), or rabbit anti-HIF-1a (1:100, Boster), then incubated with biotinylated anti-rabbit IgG (1:100, Santa Cruz) for 1 hour at 37 °C, with the avidin-biotinperoxidase complex (1:100, Vector Laboratories) for 1 hour at 37 °C. Immunoreactivity was visualized with diaminobenzidine staining and imaged under a microscope.
To explore the promoting vessel normalization effects of ORI, we performed immunofluorescence double labeling. Sections were incubated in rabbit anti-CD31 (1:200, Bioworld), with mouse anti-a-SMA (1:100, Boster) for 1 hour at 37 °C. For macrophage polarization assessment, sections were incubated in mouse anti-CD206 (1:100, Santa Cruz) for 1 hour at 37 °C. Antibodies were then used: the fluorescein 488-conjugated donkey anti-rabbit antibody (1:1000, Jackson Immunoresearch, USA), or Cy3-conjugated sheep anti-mouse antibody (1:1000, Jackson Immunoresearch). The visual data were scanned using a fluorescence microscope.

Western blotting was used to determine protein expression as previously described [34 (link)]. TA muscle tissue was lysed in RIPA lysis buffer (Biosharp, Hefei, China). After centrifugation, soluble proteins were quantified with a BCA kit (Biosharp) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were electrophoresed until sufficiently separated and then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, and then the PVDF membranes were incubated with the following primary antibodies: rabbit anti-tubulin (1:5000, Cat. #5335S, CST), rabbit anti-cleaved caspase3 (1:1000, Cat. #9664S, CST), rabbit anti-caspase3 (1:1000, Cat. #9662S, CST), rabbit anti-Bax (1:1000, Cat. #14796S, CST), rabbit anti-Bcl-2 (1:1000, Cat. #3498S, CST), rabbit anti-CD86 (1:1000, Proteintech), mouse anti-CD206 (1:1000, Santa Cruz), and rabbit anti-CD68 (1:1000, sc-20060, Santa Cruz) at 4 °C overnight. Then, the PVDF membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated IgG antibodies at room temperature for 2 h. Bands were visualized using an ECL kit (Millipore, Billerica, MA, USA). The band densities were quantified with ImageJ software (NIH).