Ab8918

CD68 and CD86 or CD68 and CD206 double immunofluorescence staining was performed to detect M1 or M2 macrophages, respectively. After consolidation for 2 weeks, tibia specimens (n = 3) were decalcified in 18% (w/v) EDTA for 3 days after fixation. Subsequently, the samples were dehydrated in 30% (w/v) sucrose, embedded in optimal cutting temperature compound, and cut into 10-μm-thick longitudinally oriented sections. After blocking with 5% (w/v) BSA for 1 h, bone sections were incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorophore-conjugated secondary antibodies (1:200; ab6785, ab6939, Abcam, UK) at 25 °C for 1 h. Nuclei were stained with DAPI. A fluorescent microscope was used for observation and image capture. For semiquantitative analysis, the ratios of CD86⁺CD68⁺/CD68⁺ cells and CD206⁺CD68⁺/CD68⁺ cells in the distraction area were calculated using Image-Pro Plus software.
The primary antibodies used in this study included anti-CD68 (1:100; ab125212, Abcam, UK), anti-CD86 (1:100; IMG-6882A, Novus, USA), and anti-CD-206 (1:100; ab8918, Abcam, UK).

Primary antibodies included anti-Sema3A (1:100, rabbit monoclonal, Abcam, ab23393), anti-CD16/32 (1:100, goat polyclonal, R&D, AF1460), anti-CD206 (1:100, mouse monoclonal, Abcam, ab8918), anti-Iba1 (1:100, rabbit monoclonal, Abcam, ab178846), and anti-P2RY12 (5 µg/ml, mouse monoclonal, Biolegend, 848001). Secondary antibodies included anti-mouse IgG, anti-goat IgG, anti-Rat IgG, and Alexa Fluor 488, 594, and 647 conjugated to anti-rabbit IgGs (1:1000, Invitrogen). Mouse retinas were fixed in 4 % paraformaldehyde and cryo-sectioned at a thickness of 10 μm. Retina cryosections and fixed primary microglia were incubated with 0.1 % Triton X-100 (Sigma-Aldrich) in PBS at room temperature for 10 min, then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were applied for 1 h at room temperature. The slides were mounted after nuclei counterstaining (Hoechst 1:2000). Images were taken by an SP-8 confocal microscope (Leica, Germany). For quantification of immunofluorescence, CD16/32⁺, CD206⁺, and Iba1⁺ cells were counted in 3 s. (10 μm thick) of retinal tissue per mouse. The mean number of cells per field was determined. All data were expressed as mean (n = 3 mice per group) ± SEM. The sections were selected randomly and were analyzed under a Leica SP8 confocal laser scanning microscope. Quantification of cell numbers was analyzed using ImageJ software.

The synovium tissue of the inferior patella was fixed with 4% paraformaldehyde for 48 h, and then embedded in paraffin after dehydration. The continuous sections with a thickness of 5 μm were prepared. The SP (Streptavidin perosidase) method was used and SP immunohistochemistry kit was purchased from Beijing boason Biotechnology Co., Ltd., (Beijing, China). In short, after conventional dewaxing, the tissue sections were incubated in 3% hydrogen peroxide in 37°C for 10 min, put into citric acid buffer (pH6.0), boiled (95°C, 15 min), and cooled for 20 min. The normal goat serum working fluid was closed in 37°C for 10 min. The anti F4/80 (1:100, ab16911, Abcam), iNOS (1:100, ab49999, Abcam) and CD206 (1:100, ab8918, Abcam) were all purchased from Abcam company. The secondary antibodies labeled with biotin were incubated overnight at 4°C and incubated at room temperature for 1 h. Then, the streptavidin labeled with horseradish peroxidase was added and incubated at 37°C for 30 min. Finally, DAB was stained for 10 min.

Whole hearts (n = 4–6 mice per treatment group) were harvested and fixed with isopentane pre-cold in liquid nitrogen for 10 s. The rapidly frozen hearts were then sectioned longitudinally at a thickness of 5 μm. Following rehydration in deionized water, tissue sections were rinsed three times in PBS (3 min). Non-specific antibody binding sites were blocked using H₂O₂ for 10 min, followed by 3% BSA for 30 min. The tissue sections were then incubated overnight at a constant temperature of 4 °C with the appropriate primary antibodies: CD68 (Abcam; Ab955), CD86(Abcam; ab53004), and CD206(Abcam; ab8918). The next day, tissues were incubated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase for 20 and 30 min, respectively. Subsequently, DAB (ORIGENE; ZLI-9018) staining and hematoxylin counterstaining were performed, followed by dehydration in ascending series of ethanol. The tissue slides were then dried in xylene and mounted for microscopy. Tissue slides were imaged at a magnification of 400 × and analyzed using ImageJ (1.52a version; National Institute of Health, United States).

Brain sections were fixed with 4% paraformaldehyde and then incubated in PBST (0.4% triton in PBS) containing 5% bovine serum albumin solution (Sigma-Aldrich, USA) for 30 min at 37°C to rupture the cell membrane and block nonspecific staining. After that, sections were incubated with primary antibodies against NeuN (1 : 1000, ab177487, Abcam), IBA1 (1 : 500, ab5076, Abcam), iNOS (1 : 300, ab15323, Abcam), and CD206 (1 : 300, ab8918, Abcam) for 24 h at 4°C followed by incubation with the corresponding secondary antibody at room temperature for 1 h [26 (link)]. After washing off unbound antibodies, the nuclei were then stained with DAPI (Biosharp, BL105A) and imaged under a fluorescence microscope (DMi8, Leica, Germany). Three brain tissue sections were randomly selected from each mouse for staining, and six microscopic fields were randomly taken around the hematoma in each section. The number of positive cells was calculated and statistically analyzed.

CD86 (Abcam; ab53004; 1:1000) and CD206 (Abcam; ab8918; 1:1000) IHC staining were done as previously described (17 (link)), but with few optimizations. Briefly, frozen sections were used; hence, the antigen retrieval step in the described experiment was skipped, and myocardial sections were fixed with 4% formaldehyde for 15 min prior to staining. Infiltrated CD86+ and CD206+ macrophages were observed at X40 magnification and quantified with ImageJ.

IHC staining for CD86 (Abcam; ab53004; 1:1000); CD206 (Abcam; ab8918; 1:1000) and IF staining for Glut1 (E4S6I) (Rabbit mAb #73,015) and F4/80(C-7): sc-377009) was performed as previously described [26 (link)], with some modifications. Frozen sections were used; therefore, the original experiment's antigen retrieval phase was skipped, and myocardial slices were fixed in 4% formaldehyde for 15 min before staining. CD86 + and CD206 + were detected using a light microscope (Olumpus CX31) at X40 magnification. Also, Glut1 and F4/80 were detected using a fluorescence microscope at X60 magnification. They were then quantified using ImageJ (1.52a version; National Institute of Health USA).