Streptavidin brilliant violet 421

Cells or frozen sections were fixed in 4% paraformaldehyde for 1 h, blocked with 5% BSA containing 0.3% Triton X-100 for 1 h, and incubated with primary antibody against CD144 (1: 500, Abcam, catalogue number: ab205336) at 4 °C overnight followed by washing 3 times with PBS and incubation with anti-rabbit IgG (H + L), F (ab ‘) 2 Fragment (Alexa Fluor® 488 Conjugate) (1: 500, CST, catalogue number: 4412S) for 2 h. After washing in PBS three times, DIPA (1 mg / ml) was added for 10 min incubation followed by washing twice with PBS, mounting on an anti-tissue fluorescence quencher and observation under a microscope.
Mice femurs were isolated and keeped in 4% PFA for 2 h at 4 °C. After decalcification and dehydration, the femurs were embedded with 8% gelatin 20% sucrose 2% PVP and kept at − 80 °C. Femurs were sliced into 20–30 μm thickness. Non-specific binding were blocked with 4%BSA in PBS at room temperature for 1 h. The desired primary antibody diluted in PBS with 1%BSA were covered the sections at 4 °C overnight. For bone marrow endothelial staining, we used Mouse Endomucin Antibody (RD, AF4666). Rat anti-CD150 (Biolegends, 115,902), Rat anti-CD48 (Biolegends, 103,432), Rat anti-lineage-biotin (Biolegends), DyLight® 550 (ThermoFisher, SA5–10027) and Streptavidin-Brilliant Violet 421™ (Biolegends, 405,225) were used for HSC staining.

Isolation of mouse satellite cells was reported previously (Uezumi et al., 2016 (link)). Hind limb muscles were collected, minced and digested with 0.2% type II collagenase (Worthington) for 60 min at 37°C using a magnetic stirrer. Digested muscles were passed through an 18-gauge needle several times and further digested for 30 min at 37°C. Digested samples were filtered through a 100-μm cell strainer, and then through a 40-μm cell strainer. Cells were resuspended in washing buffer and labeled with APC-eFluor 780-conjugated rat anti-mouse CD45 (1:250) (Invitrogen), PE/Cy7-conjugated rat anti-mouse CD31 (1:250) (Biolegend), biotin-conjugated SM/C-2.6 (1:250) (Fukada et al., 2004 (link)), and PE-conjugated goat anti-mouse PDGFRα (15 μl/test) (R&D systems), followed by secondary staining with streptavidin-brilliant violet 421 (Biolegend) (1:250). CD31^–CD45^–PDGFRα^–SM/C-2.6⁺ cells were sorted and collected as satellite cells with FACSAria II (BD Biosciences).