Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

The overall goal of this procedure is to generate three-D renderings of placental villous tree fragments that are suitable for the analysis of their contained vascular networks. This method can help us answer key questions in our field, such as why a child may be growth restricted or what stress and how severe the stress might have been that lead to pre-term birth. The main advantage of our procedure is that we are now in a position to immunolabel clear placental villous tree tissue and then visualize their vascular networks and use those visualizations to characterize and analyze these vascular networks.

The implications of this technique can extend into many diagnoses and therapies since most life sciences rely on two-dimensional histological characterization. Demonstrating the procedure will be Ms.Valerie Schwenk who will be carrying out the immunolabeling and clearing of the placental tissue and then Mr.Phillip Necaise will be doing the dissection of the placental tissue and doing also the reverse clearing of the tissue. To prepare for the dissection of the villous tree, first rinse the formal and fixed placental tissue with phosphate buffered saline.

Then place in a petri dish on the stage of a dissection microscope. To locate the villous tree, use scalpel and fine forceps to tear the placental tissue apart. Then, dissect pieces of the villous tree approximately five millimeters in length and transfer into a microtube containing one milliliter of phosphate buffered saline.

Replace the phosphate buffered saline with a fresh one and wait for 10 minutes with occasional swirling. Then, replace the phosphate buffered saline with permeabilizing buffer to permeabilize the tissue and block non-specific binding of secondary antibodies. Then, incubate for 30 minutes.

Then, replace the permeabilizing buffer with phosphate buffered saline containing two percent goat serum with mouse monoclonal anti CD31 and rabbit anti CK7. Leave the tissue at four degrees Celsius over night. The following day, remove the remaining antibodies with additional washes with phosphate buffered saline with two percent goat serum.

Then, remove the phosphate buffered saline with two percent goat serum and add the same buffer, supplemented with goat anti-mouse immunoglobulin G coupled with a green fluorophore and goat anti-rabbit immunoglobulin G coupled with an infrared fluorophore. Incubate for 30 minutes in the dark at room temperature. Then, wash the tissue once with PBS-GS and store labeled tissue in the dark at room temperature until clearing.

Wash the samples three times with PBS at 10 minute intervals. To dehydrate the tissue, remove the phosphate buffered saline and add 50%ethanol to the tissue and incubate for 10 minutes. After incubation, discard and subsequently wash three times with 70%ethanol at 10 minute intervals followed by three washes with 100%ethanol at 15 minute intervals.

Then, use fine tipped forceps to transfer the tissue to a microtube filled with one milliliter of solution one for four hours. After four hours, transfer the tissue to a microtube filled with solution two and incubate for another four hours. To mount the tissue in a Sykes-Moore Chamber use fine-tipped forceps to position a 25 millimeter round cover slip on the chamber base.

Again, use the fine-tipped forceps to place a rubber gasket in the base on the cover slip. Remove the tissue immersed in solution two and place it on the center of the cover slip. Then, add a drop of solution two, approximately 40 microliters, onto the tissue.

Then, take a second cover slip and place it on top of the gasket. Then, compress the cover slip and the gasket firmly by threading a locking ring into the top of the base. Then, insert a 25 gauge needle through the gasket into the port on the base.

Next, fill a three milliliter syringe with one point five milliliters of solution two. Then, mount a 25 gauge needle on the syringe and insert the needle in the opposite port through the gasket. Then, start injecting the chamber with solution two and check that the air inside the chamber is venting through the other 25 gauge needle.

When the chamber is full, remove the needle and syringe and place the Sykes-Moore chamber holder on the confocal microscope stage. Use a 10X objective to focus the tissue placed on the chamber. Next, set a reference point for the stage and move the microscope stage up and down through the focal plane to identify and set the top and bottom of the tissue.

Set the step size to two point five micrometers and collect a confocal image file containing a set of images of the tissue from top to bottom. Then, transfer the tissue to a microtube containing more than 20 times the volume of 100%ethanol to reverse the clearing. Then, after every one hour interval, replace with 70 and next with 50%ethanol and finally phosphate buffered saline.

Store at four degrees Celsius in the dark until embedding. Here, to show the placental vascular network, both microscopic and confocal microscopic images of a human placenta, have been acquired. These microscopic images of the placenta show the radiating villous tree that have penetrated the placental parenchyma.

These microscopic images show the presence of a distal portion of pieces of villous tree terminating in the cluster of villi. Next, to show the trophoblast layer of the villous, the distal portion of the villous tree was immunolabeled. In the image, the outer trophoblast layer is shown in green and the capillaries are shown in red.

Then, to check the placental tissue before and after clearing confocal images of uncleared and cleared placental tissues were acquired. The images show that for an uncleared tissue, immunofluorescence can be captured at 100 micrometer or lesser depth of the tissue. On the contrary, for cleared placental tissue immunofluorescence can be captured at a much higher depth.

Further, to compare the conventional tissue with the reversed clear sections, the cleared tissue was dehydrated in a reverse process with ethanol. The immunohistochemical staining of the placental tissue with anti CK7 before and after clearing demonstrate no significant alteration in the tissue morphology due to clearing. The animation shows that the immunostained villous tissue is distributed along the whole depth of the tissue volume.

Once you're comfortable doing the procedure you can expect it to take about 18 hours, split out across three days. In attempting to do this procedure it is important to remember that it requires several different skill sets. One needs to be able to do placental pathology, confocal microscopy.

You need to be able to generate three-D renderings of the imaging, and you need to be able to be conversant in immuno processing and analysis. Following this procedure, other methods like traditional histology, STS page, or maldi can be conducted. With its development, this technique paves the way in the field of maternal fetal medicine, neonatology and trophoblast biology to explore the root causes underlying pre-eclampsia, poor fetal growth and fetal distress.

After watching this video, you should have a good understanding of how to clear, render, and analyze placental villous tree tissue. Thanks so much for watching the video and good luck with your experiments.