Af647 anti mouse f4 80

For cell assays, BMMs plated on 96-well plates were washed, fixed, and permeabilized with 4% paraformaldehyde (0.3% Triton X-100). After 30 min of blocking with 1% BSA, the cells were incubated overnight at 4 °C with primary antibodies against CD206 and CD86 (1:100; Abcam, UK). On the second day, cells were incubated with secondary antibodies conjugated with AF488 and Cy3 (Abcam, UK) for 1 h at room temperature. Finally, the nucleus was stained with DAPI for 5 min.
For bone tissue assays, femurs were dissected and then fixed in 4% paraformaldehyde at 4 °C for 48 h. Subsequently, femurs were embedded in paraffin (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted June 25, 2021. ; https://doi.org/10.1101/2021.06.25.449833 doi: bioRxiv preprint after decalcification in 10% EDTA for two weeks. Samples were longitudinally cut into 4-µm-thick sections along the sagittal plane of the femur, including the metaphysis and diaphysis. Then, bone sections were double stained with AF647 anti-mouse F4/80 and AF488 anti-mouse CD206 (BioLegend, USA) overnight at 4°C while avoiding light.

For cell assays, BMMs plated on 96-well plates were washed, xed, and permeabilized with 4% paraformaldehyde (0.3% Triton X-100). After 30 min of blocking with 1% BSA, the cells were incubated overnight at 4°C with primary antibodies against CD206 and CD86 (1:100; Abcam, UK). On the second day, cells were incubated with secondary antibodies conjugated with AF488 and Cy3 (Abcam, UK) for 1 h at room temperature. Finally, the nucleus was stained with DAPI for 5 min.
For bone tissue assays, femurs were dissected and then xed in 4% paraformaldehyde at 4°C for 48 h. Subsequently, femurs were embedded in para n after decalci cation in 10% EDTA for two weeks.
Samples were longitudinally cut into 4-µm-thick sections along the sagittal plane of the femur, including the metaphysis and diaphysis. Then, bone sections were double stained with AF647 anti-mouse F4/80 and AF488 anti-mouse CD206 (BioLegend, USA) overnight at 4°C while avoiding light.