Wga alexa fluor 555

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.

For flow cytometry experiments, SI organoids were generated from crypts extracted from Lgr5-GFP mice. After 4 days of treatment, organoids were harvested and washed 5 times with cold PBS-BSA 0.1%. Organoids were disaggregated to single cells by incubating with TrypLE supplemented with DNase I for 5 min at 37 °C. Cells were incubated in Fc block (1:1000; Invitrogen, Cat. No. 14-0161-85) and Fixable Viability Dye eFluor 780 (1:1000; eBioscience, Cat. No. 65-0865-14) for 15 min at 4 °C, and subsequently stained with anti-Epcam PE-Cy7 (1:200; BioLegend, Cat. No. 118215), anti-CD24 BV421 (1:200; BioLegend, Cat. No. 101825) and WGA Alexa Fluor 555 (1:5000; Invitrogen) for 1 h at 4 °C. Flow cytometry was performed using an LSRFortessa flow cytometer (BD Biosciences, USA), and analysis was perfumed using FlowJo v10 (Treestar, USA).

Reproductive tracts of 3–4-day old females were dissected 3 h post mating (hpm) in PBS solution and fixed in PBS with 4% paraformaldehyde, washed in PBS and treated with 3% hydrogen peroxide solution (in PBS) to quench autofluorescence. Samples were washed in PBS and permeabilized for 1 h in PBS with 0.2% Triton X-100, then blocked and permeabilized over night at 4 °C in PBS with 1% BSA and 0.1% Triton X-100. Samples were incubated in 1.5 μg/ml mouse anti-5194 antibody diluted in blocking buffer, washed and then stained with anti-mouse Alexa Fluor 488 (Invitrogen). Samples were then incubated with 5 μg/ml of wheat germ agglutinin (WGA)-Alexa Fluor 555 (Invitrogen) and in 1 μg/ml of DAPI (4,6-diamidino-phenylindole, Sigma-Aldrich). All staining steps were followed by washes in PBS with 0.1% Triton X-100. Tissues were then mounted in Vectashield medium (Vector Laboratories, Inc.) and visualized using a Zeiss Axio Observer inverted fluorescent microscope with ApoTome 2. Post-imaging processing was performed with Fiji software.