E12.5 mutant and control embryos underwent cardiac perfusion with Alexa Fluor 555-conjugated 70 kDa dextran (50 μl of 2.5 mg/ml in PBS;Invitrogen) using a pulled glass pipette with mouth connector and tubing (Sigma, P0799) in the course of the 15–20 min when a visible heart beat could be observed. Fluorescent visualization of the entire vasculature confirmed the perfusion. Tracer was left circulating for 10 min at room temperature, embryos were decapitated, their heads fixed in 4% PFA overnight at 4°C, cryosectioned and stained as described above. Staining was performed with the following primary antibodies; CD31/PECAM1 (R&D systems, AF3628) 1:1000 and monoclonal TER-119 (R&D systems, MAB1125) 1:250 followed by a 2h incubation at room temperature with DyLight Fluor-coupled secondary antibodies (1:1000, Thermo Scientific Pierce).
Alexa fluor 555 conjugated 70 kda dextran
Manufactured by Thermo Fisher Scientific
Alexa Fluor 555-conjugated 70 kDa dextran is a fluorescently labeled carbohydrate polymer. It consists of a 70 kDa dextran molecule covalently linked to the Alexa Fluor 555 fluorescent dye. This product is designed for use as a tracer or marker in various biological applications.
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2 protocols using alexa fluor 555 conjugated 70 kda dextran
1
Vascular Network Visualization in Embryos
2
Vascular Perfusion Analysis in Embryos
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E12.5 mutant and control embryos underwent cardiac perfusion with Alexa Fluor 555-conjugated 70 kDa dextran (50 μL of 2.5 mg/mL in PBS; Invitrogen) using a pulled glass pipette with mouth connector and tubing (Sigma, P0799) in the course of the 15 to 20 minutes when a visible heart beat could be observed. Fluorescent visualization of the entire vasculature confirmed the perfusion. Tracer was left circulating for 10 minutes at room temperature, embryos were decapitated, their heads fixed in 4% paraformaldehyde overnight at 4°C, cryosectioned and stained as described above. Staining was performed with the following primary antibodies; CD31/PECAM1 (R&D systems, AF3628) 1:1000 and monoclonal TER-119 (R&D Systems, MAB1125) 1:250 followed by a 2 hours incubation at room temperature with DyLight Fluor-coupled secondary antibodies (1:1000, Thermo Scientific Pierce).
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