The overall goal of this procedure is to perform whole Mount immunohistochemical analysis for imaging the entire vasculature in developing limb skin. This is accomplished by first cutting four limbs from the embryo, fixing the four limbs in 4%paraform aldehyde in PBS, and then dehydrating them in 100%methanol. The second step is to use fine tweezers to peel off the skin at the base of the limb.
Follow the blue dashed lines and peel off the skin in a dish containing 100%methanol. Remove the skin from the limb. Next immunohistochemical staining with vascular specific primary antibodies and fluorescent secondary antibodies is performed on the limb skin.
Finally, whole mount confocal microscopy with multiple labeling will enable robust imaging of the entire limb skin vasculature That may advantage of this technique over existing methods such as cross count histology analysis. Using embryo sectioning is that whole mount immunohistochemical analysis of limb skin vasculature allows us not only to image the entire vasculature, including the cell components, but also to study processes of differentiation and the patterning of blood vessel cells and the lymphatic vessel branching. Are we demonstrating the procedure together with winding DA postdoc from my laboratory, Embryonic limb skins are collected from pregnant female mice.
After removing and washing the uterus, work under a dissecting microscope to separate and dissect the embryos. Once the embryos have been dissected, the next step is to remove the four LIMS from the embryos using fine tweezers. Cut the four lim at the shoulder, then use a ring forceps to transfer the four lim to a 24 well plate containing ice.
Cold, fresh 4%Paraform Hyde in PBS place two four LIMS per well in two milliliters of 4%PFA. Fix the four limbs with gentle mixing on a mutator mixer at four degrees Celsius overnight on the following day. Remove the PFA from each well and add two milliliters of PBS.
Wash the specimens for five minutes with gentle mixing on the mutator mixer at room temperature. Repeat this wash twice for a total of three washes. Next, transfer the four limbs to a five milliliter polypropylene round bottom tube containing 100%methanol store at minus 20 degrees Celsius in an enzyme freezer with critical temperature control and without an automatic defrost function After overnight storage and the minus 20, the skin can be peeled off from the falling.
This is one of the most critical aspects of this procedure because the success of the subsequent staining and imaging depends on not damaging the skins. The limb skin separates easily from the limb when dehydrated in this manner to peel off the skin from the four limb. First place the limb with the ventral side facing up.
Then using fine tweezers, dissect through the skin near the base of the limb toward the paw in a straight line. Next, dissect around the entire limb, peeling the skin off as you turn the limb. Once the skins have been peeled off.
The four limbs begin whole mount immunohistochemical staining of the limb skins Prepare beforehand. Three stock 50 milliliter tubes with 75%50%and 25%methanol in PBT for rehydrating. The skins, the limb skins are rehydrated by incubating through a grated series of methanol from the highest to the lowest methanol concentrations for five minutes each.
To begin transfer the limb skin to a five milliliter polypropylene round bottom tube containing 75%methanol in PBT. After five minutes on the mutator mixer, carefully aspirate the 75%methanol and refill with 50%methanol and PBT. Five minutes later.
Replace the 50%methanol with 25%methanol and PBT. At the end of the final incubation. Carefully aspirate the 25%methanol PBT and refill with PBT.
Wash the limb skins for five minutes with gentle mixing on the mutator mixer at room temperature. Repeat this wash once. Being careful to avoid damaging the limbs skins during the exchange of solutions.
Once the limbs skins have been rehydrated, block them with the appropriate blocking buffer. Incubate for two hours with gentle mixing on the nutt mixer at room temperature After two hours, place the limb skins on a 35 by 10 millimeter Petri dish. Then use ring forceps to transfer the skins into a two milliliter fuge tube containing 800 microliters of primary antibodies diluted in the appropriate blocking buffer.
Here, multiple primary antibodies derived from different species can be used simultaneously. Incubate the limbs skins with gentle mixing on the mutator mixer at four degrees Celsius overnight on the following day, place the limbs skins on a 35 by 10 millimeter Petri dish. Transfer the skins into a five milliliter polypropylene round bottom tube containing four liters of the appropriate washing buffer.
Wash five times for 15 minutes each with gentle mixing on the mutator mixer at room temperature. Carefully replace the used buffer with new buffer for each wash during the washes. Prepare the secondary antibodies to remove aggregated particles of the secondary antibodies.
Use a 0.22 micron PVDF membrane syringe filter to filter the secondary antibody solution into a 1.5 milliliter fuge tube. Then centrifuge the filtered sample at 15, 000 RPM for 10 minutes at four degrees Celsius to remove particles and air bubbles. Collect the filtered antibody solution in a two milliliter micro fuge tube.
When the five washes are done, place the limbs skins on a 35 by 10 millimeter Petri dish and transfer them into a two liter micro fuge tube containing 800 microliters of secondary antibodies in the blocking buffer. Different fluorescent conjugated secondary antibodies derived from different species can be used simultaneously in this step. Incubate the limbs, skins in the dark for one hour with gentle mixing on the mutator mixer at room temperature.
After the one hour incubation, place the limbs skins on a 35 by 10 millimeter Petri dish and transfer them into a five milliliter polypropylene round bottom tube containing four milliliters of the appropriate washing buffer. Wash five times for 15 minutes each in the dark with gentle mixing on the Tator mixer at room temperature for imaging by confocal microscopy. The limb skins must be mounted on a slide after immunohistochemical staining.
To begin this procedure, place the stained limb skins on a 35 by 10 millimeter Petri dish, working under a stereo microscope with low illumination. To avoid extensive photo bleaching, use fine tweezers to remove dust crystals and fibers from the inner layer of the skins. Transfer the limb skins to an adhesive microscopy slide.
Place the skins with the inner layer lying upward on the slide facing the cover slip. Then flatten the skins carefully using fine tweezers. Remove carry over washing buffer by Kim wipe.
Add Antifa mounting media to the skins and place a 25 by 25 cover slip on top of the specimens. Taking care not to introduce air bubbles cure on a flat surface in the dark at room temperature for long-term storage of the specimens. Seal the cover slip to the slide with nail polish and store at four degrees Celsius.
The patterning of nerves and blood vessels in mouse for limb skin at E 15.5 is shown by these representative results of whole mount triple label confocal immunofluorescence microscopy immunofluorescence of the pan endothelial cell marker pcam one is blue. The neurofilament marker two H three is green, and the smooth muscle cell marker alpha SMA is red. These images revealed a characteristic branching pattern of alpha SMA positive arteries indicated by white arrowhead aligned with two H three peripheral nerves indicated by open arrows in addition to blood vessel branching.
This limb skin vasculature model can also reveal the patterning of lymphatic vessel branching using the lymphatic endothelial cell marker LYVE one shown in red and neuro pill in two shown in green lymphatic vessels are visualized by both LYV one and NRP two, whereas LYV one is also expressed by a subset of macrophages indicated by the open arrow heads. This technique paves the way for researchers in the field of vascular biology to explore the complex patterning and the differentiation processes of vasculature during tissue development, tissue repairs, and in disease conditions.
