Biotin anti cd48

Endogenous HSC staining in sternal BM was performed as previously described^{11 (link)}. Briefly, Alexa Fluor 647-anti-CD144 (Biolegend, cat# 138006, clone BV13, 1:100) and Alexa Fluor 647-anti-CD31 (Biolegend, Cat# 102516, clone MEC13.3) were i.v. injected into mice to stain the vasculature. Sternal BM fragments were removed 10 min after antibody injection, fixed with 4% PFA for 30 min, rinsed with PBS for three times, and then blocked/permeabilised in PBS containing 20% normal goat serum and 0.5% Triton X-100 at room temperature. Primary antibodies were incubated for 2 overnights at room temperature. The primary antibodies used were biotin-anti-lineage (TER119, RB6-8C5, RA3-6B2, M1/70, 145-2C11, BD Biosciences, cat. No. 559971, 1:100), biotin-anti-CD48 (eBioscience, cat# 13-0481, HM48-1, 1:100), biotin-anti-CD41 (eBioscience, cat# 13-0411, MWReg30, 1:3000), PE-anti-CD150 (Biolegend, cat# 115904, clone TC15-12F12.2, 1:100). After rinsing the tissue with PBS, the tissues were incubated with streptavidin eFluor 450 (eBioscience, cat# 48-4317, 1:100).