The overall goal of this procedure is to volumetrically image fluorescent protein labeled cells deep inside intact hearts, to study gene function at single cell-resolution during cardiac development and in postnatal hearts. This method can help to answer key questions during cardiac development, such as the study of the gene functions and helping image at single-cell resolution during cardiac development and animidal artifacts. The main advantage of this method is it is relatively simple, inexpensive, and pro-wise in that special temporal analysis of the gene and protein functions at single-cell resolution in intact embryo or heart.
Although we use this system to study heart development in mice, you can also use it to study other organ systems, such as muscles, brain, and lungs and in other model organisms as well. Fluorescent protein positive embryos from Tamoxifren-treated R-26 Cre-RET2 confetti dams are used here. Begin staining by immersing each paraformaldehyde-fixed embryo in one milliliter of 0.1%PBST in the wells of a 48 well plate.
Incubate for two hours on a plate shaker at room temperature to permeabilize the tissue. Then, replace the permeabilization solution with one mL of blocking buffer and incubate, with shaking, for two hours at room temperature or overnight at four degrees celsius. Next, prepare a one to 100 dilution of the endothelial-cell specific antibody, PECAM, in 0.3 milliliters of blocking buffer for each well and add this to the wells containing the embryos.
Incubate for 48 hours with shaking at four degrees celsius. Following the incubation, wash the embryos five times with PBST for 30 minutes each time to remove excess primary antibody from the tissue. Next, immerse the embryos in Alexa Fluor 647 goat antivat secondary antibody solution and incubate with shaking for 48 hours at four degrees celsius, while protected from light.
48 hours later, after washing out before, post-fix the embryos with 4%PFA for one hour at room temperature with shaking. Then, after washing twice with PBS for five minutes each time, incubate the embryos with DAPI nuclear stain for ten minutes. Follow with two PBS washes of five minutes each, then proceed to tissue clearing.
The tissue clearing is the most critical step in this protocol in order to image the heart at a single-cell resolution. Also, the selection of the clearing method either scale or CUBIC, is also very important in order to preserve fluorescent signals in the intact embryonic heart. Transfer the washed, stained embryos into a scintillation vial wrapped in aluminum foil, that contains one milliliter of scale A2 solution.
Incubate at four degrees celsius with occasional gentle shaking by hand for 48 hours. After the scale A2 incubation, replace the solution in the vial with one milliliter of scale B4 solution, and incubate at four degrees celsius with occasional gentle shaking for another 48 hours. Incubate the embryos for another 48 hours with one milliliter of scale 70 at four degrees celsius with occasional gentle shaking as before.
Lastly, immerse the scale cleared sample in one milliliter of 90%glycerol and store at four degrees celsius until imaging. To image, transfer the sample to a glass-bottom petri dish with a minimal amount of 90%glycerol and image with a confocal microscope equipped with two photon capabilities. For CUBIC clearing, immerse each embryo in one milliliter of CUBIC one with occasional gentle shaking for 48 hours at 37 degrees celsius.
After 48 hours, replace the solution with the same volume of fresh CUBIC reagent one and incubate the sample for an additional 48 hours, as before. Following the incubation, wash the CUBIC one treated embryos with PBS several times with gentle shaking by hand to remove the excess CUBIC one solution. Then, immerse the embryos in CUBIC two solution for 48 hours at 37 degrees celsius with occasional gentle shaking by hand.
If there will be a delay before imaging, sequentially incubate the samples with one milliliter of 30%50%and 70%glycerol and PBS for 30 minutes each. After moving through the series, transfer to 90%glycerol solution for storage at four degrees celsius. When ready, image in a glass-bottom petri dish with 90%glycerol, as before.
This movie shows optical sections of a scale-treated E 8.75 heart, from heart surface to lumen. Yellow marks the PECAM expression and all the cardiomyocytes appear green. The depth is 110.4 microns and there are 93 slices in total.
This is the 3D surface picture of the reconstructed heart just shown. Again, yellow marks PECAM expression, and green labels all the cardiomyocytes. This movie shows sections of a single progenitor clone from a scale-treated E 9.5 heart in the outflow tract region.
Yellow marks the PECAM expression and the clone appears green. The depth is 36 microns and there are 10 slices in total. This movie shows sections of a CUBIC-treated P two heart from surface to lumen.
The depth is 130 microns with three microns per section. This movie shows sections of a CUBIC-treated E 17.5 heart from surface to lumen. The depth is 630 microns with six microns per section.
This is a 3D surface picture of the heart just shown, reconstructed via the 3D reconstruction software. When attempting this procedure, it is important to remember that the embryo is fragile and heart pathology can be disturbed easily. Following this procedure, other protein can be realized, using staining to study the gene function at a post-ost lation and lather.
