Wga alexa fluor 647

Frozen muscles were sectioned at 8 μm thicknesses using a Leica CM1950 cryostat maintained at −20℃. The muscle sections were first stained with hematoxylin and eosin (H/E) for histological assessment of xenograft regeneration. Muscle sections were also immunostained with human‐specific spectrin (SPEC1‐CE, 1:50, Leica) and human‐specific lamin A/C (ab40567, 1:200, Abcam), while total muscle tissue was marked by 4′6‐diamidino‐2‐phenylindole (DAPI) staining (outside human‐marked tissue) or by immunostaining with fluorescently conjugated wheat germ agglutinin (WGA alexafluor‐647, W32466, 1:500, Thermo Fisher Scientific). Specifically, muscle sections were fixed in ice‐cold methanol for 10 minutes, washed in phosphate‐buffered saline (PBS), and blocked in PBS supplemented with 2% goat serum (GTX73249, GeneTex) and mouse‐on‐mouse (M.O.M.) blocking reagent (MKB‐2213, Vector Laboratories). Muscle sections were incubated with primary antibodies overnight at 4℃ and subsequently probed with Alexa Fluor secondary antibodies (Life Technologies). Finally, muscle sections were mounted with Prolong Gold Mounting Media (Life Technologies) with DAPI for nuclear staining.

The measuring of ABCG2 levels in RBCs were carried out according to a recently developed flow cytometry-based method [25 (link), 29 (link), 30 ]. Blood samples were freshly collected (2–6 h before the flow cytometry analysis). We fixed and permeabilized the RBC membranes by using 1% formaldehyde solution, resulting in RBC “ghosts”. WGA-Alexa Fluor 647 (Thermo Fisher, cat. W32466, final cc. 1 µg/ml) were added to the fixed and washed RBC ghosts to separate them from debris during flow cytometry. Ghosts were incubated with the ABCG2-specific primary antibody (Bxp-34, mouse monoclonal antibody, Abcam, cat. ab3379) followed by a secondary, Alexa Fluor 488-labeled goat anti-mouse (H + L) antibody (Thermo Fisher, A-11001), in 96 well plates. Cellular fluorescence was measured twice each case by a FACSCanto II flow cytometer equipped with a plate loader. The agreement between the two measurements were tested by intraclass correlation coefficient (ICC). ICC estimates and their 95% confident intervals were calculated based on an absolute-agreement, 2-way model (package irr in R). Intraclass correlation coefficient (ICC) showed strong significant agreement between the two measurements of Bxp34 (ICC = 0.988, 95% confident intervals were: 0.982–0.991, p < 0.001). Therefore, we used the average of the two results for further analysis.

K562 cells were transfected by nucleofection (Amaxa Nucleofector Technology, Lonza AG) using a GPI-GFP plasmid (kind gift from D. Davis). After 24 h cells were sorted based on GFP fluorescence and co-cultured with HS-5 cells on Lab-Tek Chamber Slides (ThermoFisher Scientific) for 48 h. Additionally, both cell types were stained with WGA-Alexa Fluor 647 (ThermoFisher Scientific). Images were acquired using an SP8 Leica microscope with a ×63 objective and analyzed using ImageJ software.

To achieve triple staining of the blood vasculature, conduit channels, and lymphatic vessels in popliteal and inguinal LNs, briefly anaesthetized C57BL/6 mice were first injected with 50 μL anti-LYVE-1-Alexa Fluor 488 antibody (20 μg/mL, R&D Systems, Minneapolis, MN) into the rear right hock, an alternative injection site to the footpad, which is less invasive while allowing strong labeling [73 (link)]. After 8 h, 50 μL WGA-Alexa Fluor 647 (1 mg/mL, Invitrogen, Thermo Fisher Scientific, Waltham, MA) were injected in the same site, and after a circulation period of 1 h, the blood system of the whole body was labeled by injecting 100 μL of WGA-Alexa Fluor 555 (5 mg/mL, Invitrogen, Thermo Fisher Scientific, Waltham, MA) with 10 μL Heparin (100 units/mL) into the tail vein or vena cava of the anaesthetized mouse for a duration 2 min. Freshly excised murine tissue was fixed in 4% PFA, 3% sucrose at 4 °C overnight and embedded in LR white resin (medium grade, ProSciTech, Kirwan, Australia) for confocal imaging as described above. Standard confocal microscopy was performed using a Leica TCS SP2 equipped with a Leica HCX APO L 40.0 × 0.80 W UV water objective (Leica Microsystems, Wetzlar, Germany) at a voxel resolution of 0.36 × 0.36 × 1 μm.