Dylight 550

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.

To detect disruption of confluent TIME cell monolayers on MCs, we used the 45 kDa collagen-binding of fibronectin (FN_c) purified from human plasma (Sigma-Aldrich) conjugated to DyLight 550 with a labeling kit (ThermoFisher Scientific). The cells were grown to confluence either on gelatin-coated round 12-mm glass coverslips (GG-12-Gelatin, Neuvitro), or in spinner flasks spun at 35 rpm at 37 °C and 5 percent CO₂. On the day of the permeability assay, the cells were treated with thrombin for 30 min at 2 U/mL and with 1 μg/mL FN_cf for 5 min. The cells were washed with ice cold PBS, fixed in 4 percent paraformaldehyde/PBS for 30 min (coverslips) or in 50 percent EtOH/PBS and 1 percent FBS for 10 min at 23 °C (MCs), and washed again with PBS. The coverslips were mounted on glass slides with Prolong Gold (Invitrogen), a DAPI-containing medium. Permeability was quantified by measuring dye (DyLight 550, Ex/Em 562⁄576 nm, ThermoFisher Scientific) fluorescence amplitude per MC in epifluorescence images at 20X magnification (EVOS FL, ThermoFisher Scientific).

Wild-type ORC and Cdc6 were purified as described previously (Frigola et al., 2013 (link)). We used a N- and C-terminal protein modifications to fluorescently label ORC (-LPETGG at the C-terminus of Orc5 and Orc6, Ubiquitin-GGG-Flag at the N-terminus of Orc1), Mcm2-7 (LPETGG at the C-terminus of Mcm2, Ubiquitin-GGG-Flag at the N-terminus of Mcm4, 3xFlag-TEV[ENLYFQ/G]-GG at the N-terminus of Mcm3), and Cdt1 (Ubiquitin-GGG tag at the N-terminus, LPETGG tag at the C-terminus). The ubiquitin (in vivo) and 3xFlag-TEV (using TEV protease, NEB) fusions were removed to reveal three N-terminal glycines required for N-terminal sortase-mediated labeling. The peptides NH₂-CHHHHHHHHHLPETGG-COOH and NH₂-GGGHHHHHHHHHHC-COOH were used for N- and C-terminal labeling, respectively, and will be referred to as the N-peptide and C-peptide. The N- and C-peptides were labeled with maleimide-derivatized DY549P1 (Dyomics), DY649P1 (Dyomics), Dylight550 (Thermo-scientific), Dylight650 (Thermo-scientific), or Cy3B (Cytiva) via cystine–maleimide conjugation as described previously (Ticau et al., 2015 (link)). Sortase was used to couple the fluorescently labeled peptide to the N- or C-terminus of the helicase-loading proteins as described below. The peptide-coupled proteins were separated from uncoupled proteins using Ni-NTA Agarose (Qiagen).