The overall goal of the following experiment is to determine the effects of specific genetic mutations on vascular development and structure. This is achieved by dissecting the embryo and extra embryonic tissues together to provide an appropriate sample for injection. Next, the facial vein is injected with a fluorescent dye to label the intact vasculature.
Then the embryo is prepared for analysis. Finally, hole mounts or sections of labeled vasculature a image to show the three dimensional structure of the normal vasculature based on immunofluorescence microscopy of hole mount or tissue sections. I first had the idea for this method when I was interested in determining the developmental time point that the peripheral vasculature connects to the thymus via angiogenesis.
To begin prepare FXI dextran or GS L one I selectin B four in PBS in a 1.5 milliliter einor tube add 100 microliters of stock 1.25 millimolar fast green PBS to their FTSE dextran, or 180 microliters to the GS L one. I select B four so that the solutions are visibly blue. Next, warm the tube to 37 degrees Celsius.
Dissect E 14.5 to E 18.5 embryos and the yolk sacks together, leaving the Allen Toic stalk intact. Transfer the embryos to a new Petri dish and immerse them in PBS at room temperature. Next position an embryo to obtain a sagittal view of the face.
Then with micro dissecting forceps, gently grasp the embryo at the head. Using a 30 gauge needle inject 50 microliters of FXI dextran or GS L one I selectin B four into the facial vein, pointing the needle toward the back of the head. When the dye is visible in the umbilical vein, remove the needle and separate the embryo from the lan.
Toic stor. Allow the embryo to remain in PBS at room temperature for two to three minutes so that the dye circulates throughout the embryo. After allowing the dye to circulate throughout the embryo, remove skin samples from regions of the limbs back and stomach.
Wash the skin samples in cold PBS and fix them in 4%paraldehyde PBS for two hours. Then wash them three times for 10 minutes each in four milliliter clear vials with two milliliters of cold PBS. Place the skin samples on microscope slides and add 100 microliters of mounting medium and a cover slip to each slide.
Allow the slides to dry in the dark before imaging clearing in BABB and subsequent confocal imaging can be performed for higher resolution images of the skin vasculature. To prepare tissues for cryosectioning flash freeze whole embryos in liquid nitrogen for cryosectioning. Spread OCT on a section block and mount the embryo.
Cut frozen tissue into 10 micron thick sections and collect on slides. Fix the sections in acetone for five to 10 minutes, then wash them three times in cold TBS block in 10%Donkey serum in TBS in a humidity chamber. At room temperature, add 100 microliters of primary antibody to each section.
In this example we use rat anti-US CD 31 to label endothelium and goat anti-US P-D-G-F-R beta to label perivascular cells to ensure that the antibody is uniformly spread across the section. Cover slides with individually cut para film strips. Incubate the slides for one hour to overnight in a humidity chamber at four degrees Celsius.
Next, wash the sections three times in cold. TBS incubate with 100 microliters of the appropriate secondary antibodies for a minimum of 30 minutes. Finally, wash three times in cold TBS Add 100 microliters of mounting medium and a cover glass to each slide.
Allow the slides to dry in the dark. For vibram sectioning, dissect out the thymus lobes from embryos and rinse in cold PBS. Fix the tissue in 4%paraldehyde PBS at room temperature for two hours.
After washing the samples in PBS Triton X, fill a small plastic cartridge with 4%low melt arose. PBS submerge the samples in the arose PBS, making sure that the tissue is in contact with the bottom of the cartridge. Allow the arose to solidify on ice.
Then use a razor blade to cut off the excess agros. Apply glue to the VIBRAM block and a adhere the sample to the block. Pour cold PBS into the Vibram water bath until the sample and blade are immersed.
Set the speed and amplitude. The amplitude should be reduced. If sections break up due to excess agitation, cut 50 sections of tissue.
Then using forceps. Transfer the sections into cold PBS in a 24 well microplate block the sections in 500 microliters of 10%donkey serum in PBS Triton X for 30 minutes after adding primary antibodies such as anti CD 31 and anti P-D-G-F-R beta incubate for eight hours to overnight in a covered microplate of four degrees Celsius. Next wash three times in PBS Triton X over a total of eight hours at four degrees Celsius.
After blocking the sections again and incubating in secondary antibodies, wash the samples three times in PBS Triton X over a total of eight hours at four degrees Celsius. Then re-fix the samples in 4%paraldehyde PBS for 30 minutes on ice and wash three more times in PBS Triton X over 30 minutes on ice. Dehydrate the samples through a graded methanol PBS Triton X Series for 10 minutes at each step.
Then replace the 100%methanol with fresh methanol and incubate for one hour at room temperature. Place the sample in a glass depression slide and cover the sample with a one-to-one solution of BABB and methanol. Incubate the samples for 10 to 15 minutes.
Next, replace the solution with 100%BABB and incubate at room temperature for 10 to 15 minutes or until cleared. Add a cover glass and seal with two to three coats of nail polish. Allow the nail polish to harden in the dark of room temperature.
The sample can be stored at four degrees Celsius, but you should take images within 12 to 24 hours. Image 10 micron frozen sections with a confocal microscope using the plan A per chromat 20 times 0.8 objective with 6 33 4 88 and 5 43 nanometer laser lines acquire one micron confocal Z sections of 50 micron aros embedded sections using the plan A per chromat 10 x 0.4 with 4 88 5 43 and 6 33 nanometer laser lines reconstruct serial Z sections using Zeiss AIA vision 4.6 or rather image analysis software efficient labeling of the embryonic vasculature is critical for assessing blood vessel defects in embryonic mice. These figures show specific labeling of E 16.5 thymus blood vessels and co labeling of a GS L one I selectin.
B four injected embryo with CD 31 as well as staining of the right and left ventricles. The GSL one I selectin B four protocol for cryo sections was used in these experiments. Hole mount labeling of the skin blood vessel network on E 16.5 mice is shown here Following this procedure.
Other methods like co labeling of the stromal cells and blood cells with the vasculature can be used in order to answer additional questions like How do blood cells and stromal cells interact with the vasculature in the mouse embryo under both normal and pathological conditions?
