The overall goal of this procedure is to enable analysis of histological and molecular parameters from adjacent sections of a single tissue. With this method, we can help to answer key questions in the field skeletal muscle biology and disease. Such as, what are the regenerative effects after muscular injury, and what are the effects of viral gene delivery by intramuscular injection?
The advantage of this method is that histological and molecular data can be directly correlated from a single tissue. That makes more effective data analysis and better use of experimental tissues. Five minutes prior to completing the dissections, cool the cryopreservation bath.
When the tissue samples are ready to be embedded, first apply embedding resin to the lower third to half of the tissue, where the muscle meets the cork. This should be done neatly to reduce resin interference with the downstream steps. Then, immediately drop the cork into the 2-Methylbutane bath cooled to negative 140 degrees Celsius.
Include up to eight tissue preparations per batch. In the bath, stir the tissues for 30 seconds, scraping the bottom of the beaker to prevent the tissue corks from freezing into the solution. After 30 seconds, using a metal tool, pull a tissue cork from the 2-Methylbutane.
Then, quickly remove the supportive cover slip. Dab the excess 2-Methylbutane back into the bath, and then drop the tissue cork into the outer nitrogen bath. After all the tissues have been transferred into the liquid nitrogen, move them to a container and store at negative 80 degrees Celsius.
After preparing the cryostat and mounting a tissue sample, use the coarse and fine controls to advance the specimen up to the blade so it touches the blade. Reset the sum of the section thicknesses to zero at this point. Next, set the cryostat to the trim function with section thickness at 30 microns.
And cut and discard sections up to the desired tissue depth. To efficiently collect cryosections for RNA extraction, first open the collection tube and place it near the blade carrier. There, use a pre-cooled, clean brush to pick up each section and transfer it to the tube.
Pooled 30-micron mouse tibialis anterior sections taken from 400 to 4, 000 micron tissue depth typically yield 25 to 50 milligrams of muscle. If embedding resin surrounds the cryosection, lock the hand-brake and use a razor blade to shave off small pieces of resin until there is only a thin layer around the top of the muscle. Always cut resin with the blade angled away from the muscle.
A thin layer of embedding resin does not substantially impair the downstream RNA extraction. If thicker embedding resin is present, use brushes to tease it away from the muscle before moving the cryosection into the collection tube. During the sectioning process, when histological sections are desired, reset the slice thickness to seven microns using the cryostat controls.
Then, cut and discard four to seven sections to obtain a consistent, even tissue surface and make note of this tissue depth. Now, cut a good section and orient it on the surface of the blade carrier. Using a room-temperature microscope slide, pick up the section and return the slide to room temperature.
Collect all the desired sections and then make note of the final tissue depth. After completing the sectioning, quickly record the weight of the pooled cryosections for RNA isolation. Immediately return the tube to the cryostat chamber to maintain the temperature of the sections near negative 20 degrees Celsius.
Begin by quickly transferring the pooled cryosections to ice, and adding approximately one millileter of organic RNA extraction reagent per 50 milligrams of cryosections. The sooner the reagent is applied, the less the RNA degrades. Next using a one milliliter syringe with an 18-gauge needle, homogenize the cryosections without making too many bubbles by repeatedly drawing up a solution, and ejecting it onto the tube walls.
Use the needle tip to disperse clumped pieces. After every five strokes, briefly return the sample to the ice to keep it cool. Once homogenized, use a 22-gauge needle to carefully triturate the sample using five more strokes.
Then return the sample to ice. Repeat this process three or four times to achieve a very finely dispersed tissue homogeny. Once the final homogenate is made, let it incubate for five minutes at room temperature to disrupt molecular complexes.
Next, in a fume hood, add 0.1 milliliter of BCP per milliliter of RNA isolation reagent. Then, shake the tube vigorously for 15 seconds. Do not use a vortex.
Then, incubate the sample at room temperature for two to three minutes. Following the brief incubation, centrifuge the sample for 15 minutes at 12, 000g and four degrees Celsius. Then, carefully collect the clear upper phase containing the RNA.
Transfer it to a clean tube, and add an equal volume of 70%ethanol, made up in deep seawater. After briefly vortexing the tube, let it incubate for 10 minutes at room temperature and proceed by following the text protocol. Skeletal muscle RNA preparations were made from the mouse tibialis anterior.
Three days before, some muscles were injected with 10 micromolar cardiotoxin and others with saline. The purity and yield of the RNA from these samples was excellent. RNA yield was substantially higher in tibialis anterior muscles after toxin injury.
Tissue integrity and RNA production could factor into this observation. The persistence of the prepared RNA quality was assessed using one microliter of RNA that had been stored at negative 20 degrees Celsius for 18 months. Prominent 18S and 28S ribosomal RNA bands are still evident in samples demonstrating high RNA quality.
Seven-micron sections were stained for expression of embryonic myosin heavy chain, seen in red. To detect regenerating fibers, stained for collagen six, seen in green, which outlines muscle fibers and stained with dappy, in blue, to observe the nuclei. When there is a poor toxin injection, the injury appears minimal.
A successful toxin injection results in a much larger region of the targeted muscle compartment being affected. The degree of injury could be used as inclusion criteria for the RNA analysis. Once mastered, cutting sections for RNA extraction and histological slide, can be completed in under half an hour for a single tissue.
Following this procedure, other histological of immunofluorescent stains can be followed to answer a wide variety of questions. Pooled cryosections can also be used to collect DNA or protein.
