Anti cd43

Single cell suspensions were prepared and washed in ISCOVES medium. After centrifugation, erythrocytes were lysed in Red Cell Removal Buffer (RCRB; 156 mM NH₄Cl, 10 μM EDTA, 1 mM Na₂CO₃) and FCS was subsequently added (3 (link)). Cells were counted and 10⁶ cells per sample were used for staining. Cells were washed twice in PBS containing 3% FCS and 0.1% NaN₃ and were subsequently stained with optimal concentrations of anti-IgM, anti-IgD, anti-B220, anti-CD25, anti-CD21, anti-CD43, anti-Thy 1.2, anti-NK1.1, or anti-CD138 (all from BD Bioscience). Fluorochrome-labeled streptavidin and the apoptosis marker merocyanine (Sigma Aldrich, Munich, Germany) were incubated separately. Samples were subsequently acquired on a FACSCalibur (BD Bioscience) and analyzed using the CellQuest software (BD Bioscience).

Preparation of single-cell suspensions of lymphoid organs and lysis of red blood cells were performed according to standard procedures. Cells were (in)directly stained in flow cytometry buffer (PBS, supplemented with 0.25% BSA, 0.5 mM EDTA and 0.05% sodium azide) using the following fluorochrome or biotin-conjugated monoclonal antibodies or reagents: anti-B220 (RA3-6B2), anti-CD19 (ID3), anti-CD5 (53-7.3), anti-CD43 (R2/60), anti-CD23 (B3B4) all from eBioscience and anti-CD138 (281-2), anti-CD95 (Jo2), anti-IgD (11-26), anti-IgMb (AF6-78), anti-IgMa (DS-1), anti-Igλ (R26-46), anti-Igκ (187.1), anti-CD21 (7G6), all from BD biosciences, using conjugated streptavidin (eBioscience) as a second step for biotin-conjugated antibodies.
Leukemic cells (CD19⁺CD5⁺) were stained with fluorescein-labeled phosphatidylcholine (PtC) liposomes (DOPC/CHOL 55:45, Formumax Scientific Inc.) in flow cytometry buffer. Cells were co-stained with anti-CD19, anti-CD43, or anti-CD5 (BD Biosciences).

Confocal immunofluorescence analysis was performed to detect colocalization of different proteins in the endothelium of coronary arteries as we previously described 36. Briefly, the mouse hearts were frozen in Tissue‐Tek OCT and cut by cryostat into 10 μm sections and mounted on Superfrost/Plus slides. After fixation with acetone, the frozen section slides were incubated with following primary antibodies: rabbit anti‐ZO‐1 (1:100; Invitrogen), anti‐ZO‐2 (1:100; Invitrogen), anti‐Occludin (1:100; Abcam), anti‐VE‐Cadherin (1:100; Abcam), or anti‐HMGB1 (1:500; Abcam), or anti‐CD43 (1:100; BD, East Rutherford, NJ, USA). Endothelium was also visualized by costaining slides with sheep anti‐von Willebrand factor (vWF) against endothelial cell marker vWF (1:200; Abcam). After incubation with primary antibodies, the slides were washed and labelled with corresponding Alexa Fluor‐488 and Alexa Fluor‐555 conjugated secondary antibodies (Invitrogen). Then, the slides were washed, mounted and visualized through sequentially scanning on an Olympus laser scanning confocal microscope (Fluoview FV1000; Olympus). Colocalization was analysed by Image Pro Plus software, and the colocalization coefficient was represented by Pearson's correlation coefficient as previously reported 37.

The dissociated cells were incubated with antibody madoptixture at 4 °C for 30 min, followed by incubation with 7-AAD antibody at room temperature for 5 min. Cells were sorted and analyzed by flow cytometers FACS Aria II (BD Biosciences) and MoFlo XDP (Beckman Coulter) in the purity model. The FACS data were analyzed with FlowJo software (v10, Tree star). Surface markers for E10.0 early AECs in AGM regions were CD41⁻CD43⁻CD45⁻CD31⁺CD44⁺Kit⁻. Surface markers for E10.0 HECs in AGM regions were CD41⁻CD43⁻CD45⁻CD31⁺CD44⁺Kit⁺CD201⁺. Surface markers of E11.0 AGM pre-HSCs were CD31⁺Kit⁺CD201⁺. Surface markers for E14.5 fetal liver LT-HSCs were CD45⁺CD201⁺CD150⁺CD48⁻. Cells were stained using the following antibodies (1:100 diluted): Anti-CD31 (BD, MEC13.3, Catalog# 562939), Anti-CD41 (BD, MWReg30, Catalog# 553848), Anti-CD43 (BD, S7, Catalog# 553270), Anti-CD44 (BioLegend, Catalog# 103044), Anti-CD45 (BD, 30-F11, Catalog# 553079), Anti-CD48 (BioLegend, Catalog# 103432), Anti-CD201 (eBioscience, eBio1560, Catalog# 17-2012-82), Anti-Kit (eBioscience, 2B8, Catalog# 14-1171-82), and Anti-CD150 (BioLegend, Cat# 115904). 7-aminoactinomycin D (7-AAD; eBioscience, Catalog# 00-6993-50) was used to exclude dead cells. The FACS Diva 8 “index sorting” function was activated and sorting was performed in the single-cell mode.