To identify blood and lymphatic vessels during culture, tissues were incubated with 1:40 FITC-conjugated BS-I lectin (Sigma Aldrich), as previously described.8 (link),9 (link) Tissues were labeled with lectin for imaging at Day 0 and Day 2 or Day 5. Whole tissues were then mounted on glass slides, fixed in 100% methanol, and washed and permeabilized with 0.1% saponin in PBS. At the designated endpoint, tissues were stained with 1:200 biotinylated anti-rat PECAM antibody (BD Pharmingen, San Diego, CA) and 1:500 Cy2-conjugated streptavidin (Jackson ImmunoResearch) to identify endothelial cells and with 1:3000 DAPI (Life Technologies) to identify cell nuclei.
Protocol detail
Visualizing Blood and Lymphatic Vessels
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Anti antibody
Blood
Cell nuclei
Dapi
Endothelial cells
Fitc
Lectin
Lymphatic vessels
Methanol
Saponin
Streptavidin
Tissues
Corresponding Organization :
Other organizations : Tulane University, University of New Orleans
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Variable analysis
independent variables
- Incubation time (Day 0 and Day 2 or Day 5)
- Blood and lymphatic vessel identification
- Endothelial cell identification
- Cell nuclei identification
- Tissue samples
- FITC-conjugated BS-I lectin concentration (1:40)
- Biotinylated anti-rat PECAM antibody concentration (1:200)
- Cy2-conjugated streptavidin concentration (1:500)
- DAPI concentration (1:3000)
- Fixation method (100% methanol)
- Permeabilization method (0.1% saponin in PBS)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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