Generation of Porcine Ventricular Slices: A Vibratome-Based Technique to Prepare Ultra-Thin Slices of Pig Heart Tissue
내레이션 대본
To obtain pig heart tissue slices, first, add ice to the tissue bath cooling jacket of a vibratome, and add Tyrode's solution to the bath. Use a 1-liter plastic jar to collect the melted ice runoff from the tissue bath cooling jacket, and transfer the pig hearts to a tray containing 1 liter of fresh, cold, cardioplegic solution in a biosafety cabinet.
Use retractable sterile scalpels to dissect the heart to isolate the left ventricle, and use rectangular razor blades to cut the left ventricle into 1 to 2 cubed centimeter blocks. Set aside one tissue block for slicing, and place the remaining tissue block pieces into a 50-milliliter tube of cold Tyrode's solution on ice.
Gently massage the reserved tissue block, and add 1 to 2 drops of tissue glue to the metal sample holder of the vibratome. Stick a piece of 4% agar block with a surface area of approximately 1 squared centimeter to the glue, and add 1 to 2 drops of tissue glue to the agar.
Attach the heart block to the agar, cardiac epicardium side down, with the tissue as flat against the holder as possible, and place the tissue holder with the heart block into the slicing bath of the vibratome.
It is crucial to stick the heart block to the agar with the cardiac epicardium side facing down on the tissue glue, and to make sure the tissue is as flat as possible.
Then, attach the oxygen tube to the slicing bath and the metal tray filled with Tyrode's solution, and place 40-micrometer cell strainers onto the metal tray to collect the heart tissue sections as they're sliced. Use the vibratome operating software to adjust the height of the blade and sample, so that the blade is close to the top of the tissue, but below the papillary muscles, and liquid is covering the tissue and the blade.
Select Advance to adjust where to begin slicing the tissue. Press Slice to increase the speed, and use the knob to move the blade toward the edge of the tissue. Press Slice again to stop, and Advance to inactivate the process. Then, adjust the cutting parameters as indicated, and press Slice to begin slicing the tissue. When the blade reaches the end of the tissue, but before it hits the end of the specimen holder, press Slice again to stop, and press Return to go back to the start position.
When the slices reach full length and appear to be of good quality, use a plastic Pasteur pipette filled with cold Tyrode's solution to gently collect the tissue from the bath, using forceps and spring scissors to dislocate the slice from the heart as necessary. Place each collected slice in a single cell strainer in the oxygenated Tyrode's bath, and use the solution in the pipette to press the tissue on the cell strainer surface.
