At 2 h after Cy5.5-HA10K injection, popliteal LNs were harvested and were sectioned into 10 μm slices by using an Ultrapro 5000 cryostat (Vibratome). Slices were fixed with Z-fix solution for 15 min and then were blocked with PBS containing 1% BSA for 1 h. Slices were incubated with rat anti-mouse LYVE-1 primary antibody (1:400, MBL International Corporation) at room temperature for 2 h, and then with Cy3-conjugated donkey anti-rat secondary antibody (1:200; Jackson ImmunoResearch Laboratories). Between each step, the slices were gently washed 5 times with 0.05% tween 20 PBS solution (PBST) for 5 min each time. The slices were then incubated with medium containing 4′,6-diamidino-2-phenylindole (DAPI). The staining observation was done with an epifluorescence microscope (X81; Olympus). H&E staining was performed as previously reported 27 (link).
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Lymphatic Vessel Imaging in Popliteal Lymph Nodes
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Other organizations : Zhongda Hospital Southeast University, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health
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Variable analysis
independent variables
- Time after Cy5.5-HA10K injection (2 h)
- Presence and localization of Cy5.5 fluorescence in popliteal lymph node sections
- Expression of LYVE-1 in popliteal lymph node sections
- Cryosectioning of popliteal lymph node samples into 10 μm slices
- Fixation of slices with Z-fix solution for 15 min
- Blocking of slices with PBS containing 1% BSA for 1 h
- Incubation of slices with rat anti-mouse LYVE-1 primary antibody (1:400) for 2 h at room temperature
- Incubation of slices with Cy3-conjugated donkey anti-rat secondary antibody (1:200)
- Washing of slices 5 times with 0.05% tween 20 PBS solution (PBST) for 5 min each time between steps
- Incubation of slices with medium containing 4′,6-diamidino-2-phenylindole (DAPI)
- Observation of staining with an epifluorescence microscope (X81; Olympus)
- H&E staining as previously reported in the referenced publication
- Not explicitly mentioned
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