This protocol standardizes new tissue analysis techniques so that they can be more widely used by other groups who also study congenital heart defects. As both qualitative and quantitative analysis techniques have their pros and cons, this methodology uses both to address the widest range of experimental questions. These techniques can have a positive impact on cardiology diagnostic methods.
For example, pathologists could apply our microscopic cardiac tissue analysis techniques towards diagnosing human heart disease from biopsy samples. This protocol can be used for developmental cardiology or general cardiology research. However, these methods also provide various useful techniques that can be applied towards any tissue analysis.
To collect embryonic day 15 to 15.5 hearts, secure the limbs of a pregnant dam and starting around the urethra up to the sternum, carefully make an I-shaped incision along the torso. Using forceps in a scooping motion, gently lift the uterus out of the abdominal cavity grasping the superficial tissues of the uterus as necessary with fine forceps to aid in the extraction. Use scissors to release the uterus from the abdomen.
After soaking the uterus in ice-cold PBS, pin the organ into a dissection dish. Using scissors, cut the uterus into individual sections each containing a separate embryo. Using two pairs of fine forceps, gently peel the embryos out of the uterine tissue and place them in to a new Petri dish containing four degrees Celsius PBS-EDTA.
To collect the hearts, place the dish under a dissecting microscope and use two pairs of fine forceps to position the embryo into the supine position. After stabilizing access to the chest, use the sharp point of a second pair of fine forceps to make an incision along the sternum of the embryo extending between the clavicles to the navel. Making very fine and superficial incisions while gradually working toward the inside of the chest cavity, open the abdomen of the embryo.
When the heart just becomes visible, use a pair of forceps to squeeze the torso slightly coddled to the rib cage to peel the ribs open. Using the sides of one pair of forceps, gently grab the pulmonary blood vessels without separating the tissue and pull the heart out of the cavity. Clean the heart in fresh PBS-EDTA for one to two minutes before fixing the tissue in 4%Paraformaldehyde for 45 to 60 minutes.
Then, wash the heart three times for 5 to 10 minutes per wash in PBS supplemented with glycine. Before storing the hearts in PBS at four degrees Celsius. To prepare the heart for cryosectioning, place the samples into a tube of 30%sucrose and PBS at four degrees Celsius for 24 to 40 hours.
When the hearts have sunk to the bottom of the tube, use a Pasteur Pipette with a modified tip to carefully transfer each heart unto a piece of lint free wipe. After a brief air-drying, place each sample into individual optimal cutting temperature molds in the desired orientations for sectioning. Submerge the tissues in optimal cutting temperature medium without bubbles.
Then, store the molds at 20 degrees Celsius for up to a few days before freezing the tissues at 80 degrees Celsius for at least 24 hours before cryosectioning. To obtain cryostat sections of the samples, use fresh optimal temperature cutting medium to attach the tissue block of interest to the chuck of the cryostat and allow the medium to freeze until it is opaque white. Insert a new blade and adjust the distance between the tissue block and the blade to allow sections to be obtained.
Trim the block in 10 micrometer slices until the point of interest is reached. When the tissue can be observed, use a small flat paintbrush to gently pull the slice against the stand in a continuous cutting motion. Once the tissue has been completely cut through, use the detail brush to pat the slice down flat against the stage.
Hold the section in place for approximately 20 to 30 seconds before removing the detailed brush and finishing the slice. Use both paintbrushes to gently flatten the slice against the stage preventing any rolling and hold the tissue section against the stage. After 20 to 30 seconds, flip the section and flatten it against the stage again before placing an electrostatically charged slide close enough for the slice to be attracted and adhere to the slide without having to touch the slide to the stage.
To evaluate the heart tissue sections for the presence of common heart defects, using a microscope equipped with a camera, obtain images at 5 to 10X and 40X magnifications. For cardiac muscle compaction analysis, open a tissue of interest in an appropriate image analysis software program and set the image to 8-bit. To set the threshold of the image, click Image, Adjust, and Threshold and move the top bar to adjust the threshold until only the pixels for the background are selected.
Next, use the polygon selections tool to draw a region of interest around the tissue then click Analyze, Set Measurements, Area, and Limit to threshold. Click Analyze and Measure to measure the area of the highlighted pixels to obtain the value of the area of the negative space. To measure the area of the entire region of interest, click Analyze, Set Measurements, and deselect Limit to threshold.
Then, select Analyze and Measure to measure the entire area of the selected region of interest. To calculate the Area of muscle tissue, subtract the Area of Negative Space from the Area of the Region of Interest. Then divide the Area of muscle tissue by the Area of the Region of Interest to calculate the muscle compaction and X.The staining of tissue slices from harvested embryonic hearts, provides enough contrast to make out the tissue edges, but is not so dark as to make the cell features indistinguishable.
In this representative analysis, the muscle compaction was observed to be significantly reduced in the experimental hearts relative to the embryos that developed under non-experimental conditions. These samples are valuable and require a long time to create. During necropsies and the cryostat sectioning specifically, make sure to be intentional about every action you perform.
Preserving the tissues in Paraformaldehyde maintains the antigen activity permitting the samples to be used for tests like Aminofluorescein staining which help identify pathological causes found during the morphology assessment. In our lab, we obtained quantitative representation of experimental treatments which can help us to identify the degree to which different treatments affect our samples. Paraformaldehyde can fix any tissue that it touches.
Take care to always wear gloves to minimize exposed skin and to work in a film hood whenever working with this chemical.
