Anti f4 80 alexa fluor 488 bm8

Freshly isolated femurs from PDGFRα‐lineage tracing mice were prepared into frozen tissue block as previously described [53 (link)] except the fixation was extended to overnight. Sixty‐micron thick cryosections were prepared on slides. After rehydration in PBS, the tissue was permeabilized in 0.5% Triton‐X‐100 in PBS for 15 min and then blocked in 3% BSA and 0.3% Triton‐X‐100 in PBS (diluent) for 1 h at room temperature. Tissues were stained with 10 μg/mL each of anti‐F4/80‐Alexa‐Fluor‐488 (BM8), anti‐PECAM‐1‐Alexa‐Fluor‐647 (390 and Mec13.3), and anti‐Sca1‐Brilliant‐Violet‐421 (D7) (all from Biolegend) in diluent at 4°C in the dark overnight. The slides were then washed and mounted for image stack acquisition by LSM880 (Zeiss). Fiji Image J was used for 3D‐stack examination of adherent monocytes on endothelium. Gamma (0.85) was applied to the tdTomato channel for simultaneous visualization of bright perivascular cells and dimmer PDGFRα‐lineage leukocytes in BM. For examination of AD skin, samples were process similarly as described except cryosections were prepared in 100 μm‐thick and stained with 10 μg/mL each of anti‐F4/80‐Alexa‐Fluor‐488 (BM8) and anti‐PECAM‐1‐Alexa‐Fluor‐647 (Mec13.3) (both from Biolegend).