One significant barrier in studying intramuscular fat, a hallmark of sarcopenia and neuromuscular diseases, is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. The protocol preserves adipocyte morphology and skeletal muscle sections aligned for robust, rigorous and reproducible visualization and quantification of intramuscular fat. Our analysis pipeline provides a framework to validate novel interventions to prevent fat formation in diseases such as Duchenne muscular dystrophy Demonstrating the procedure will be Connor Johnson, a research technician from my laboratory.
To begin, clean the leg to be injected with a fresh alcohol wipe to disinfect. Draw up 30 to 50 microliters of 50%glycerol into the insulin syringe. Gently brush up the hair on the shin to expose the location of the TA.After locating the TA, insert the needle into the TA distally near the ankle.
Fully insert the needle into the muscle and slowly inject glycerol, while gradually withdrawing the needle which helps to injure most of the muscle. Liberally spray any areas of the mouse to be cut into with 70%ethanol to help keep hair off dissecting area and instruments. Use scissors to cut the skin around the top of the leg near the pelvis.
Gently pull the skin of the leg from the top down to the ankle. First, remove the outer connective tissue layer using sharp tip tweezers prior to harvesting the TA.Slide the tweezers underneath the TA from the bottom of the muscle, starting at the distal tendon and gently pull upward towards the knee. Stop at the end of the muscle.
Do not push past the resistance felt at the lower knee. If there is a significant resistance prior to reaching the lower knee, stop and continue removing leftover layers of connective tissue. Once the TA has been partially lifted from the leg with the tweezers, use the same motion with a scalpel to sever the connection of the TA to the lower knee.
Gut the tendon at the ankle with scissors to fully remove the TA.Only handle the muscle at the tendon to avoid damaging the fibers. Cut one third of the TA at the end, opposite the tendon. Put it in the micro centrifuge tube and snap freeze it by dropping it into liquid nitrogen.
Submerge the other two third of the tissue in a labeled well with 4%PFA for histology. Be sure to keep track of when the first and last tissue was placed in fixative. Place on a nutator for two to two and a half hours at four degrees Celsius.
After fixation, remove PFA from the wells. Rinse the tissues with cold one X PBS two to three times and then wash two to three times with cold 1X PBS for five minutes per wash. Remove the PBS from the wells and add enough 30%sucrose in 1X PBS to allow the tissue to float.
Place on the nutator at four degrees Celsius overnight. Turn on the microscope and launch the imaging software. Secure the slide on the stage.
Use any channel to identify the area to be imaged. In the imaging software, adjust the gain and exposure time for each channel. Take images of the whole tissue in each channel and merge individual tiles to make a composite of the full TA cross section.
Before beginning, preheat RNase free water to 45 degrees Celsius and prepare fresh 70%ethanol. Add 1000 microliters of guanidinium thiocyanate to each tube containing the sample. It's important that Bead Beater approved tubes are being used.
Add three medium beads, or one large and one small bead, to each tube. Homogenize the tissue at 50 Hertz for two to four minutes using a Bead Beater. Depending on tissue type and sample size, it may take up to 10 minutes.
add 200 microliters of chloroform. Shake the samples for 15 seconds. Incubate for two to three minutes at room temperature.
Centrifuge for 15 minutes at 12, 000 times G.Pipette out 350 microliters of the clear supernatant and add to a new micro centrifuge tube containing 350 microliters of 70%ethanol. Be careful not to aspirate the lower protein and/or DNA layers. Transfer up to 700 microliters of this mixture to a mini spin column placed in a two milliliter collection tube.
Continue with RNA isolation following the manufacturer's instructions. Elute with 30 to 50 microliters of RNase free water, depending on expected yield. Keep RNA on ice and measure the yield using a spectrophotometer.
Keep the RNA stored at minus 80 degrees Celsius. Use up to one microgram of RNA to synthesize cDNA with a cDNA synthesis kit, following the manufacturer's instructions. After the run is complete, add 80 microliters of RNase free water.
Store the samples at minus 20 degrees Celsius. Using a 384 well format, add one microliter of primer to the bottom of each well. Free drying primers result in tighter technical replicates.
Leave covered until primers have evaporated completely. Set up sample reactions with four to eight technical replicates. After the PCR run is complete, normalize raw cycle threshold values to housekeeping gene levels and determine fold changes by calculating delta-delta Ct.Perilipin and positive adipocytes from the unfixed TAs have significantly altered morphology compared to fixed sections, making their identification, visualization, and a subsequent quantification much more difficult and potentially inaccurate.
In contrast, PFA fixation preserves adipocyte morphology, allowing for accurate detection of intramuscular fat. Compared to uninjured TA muscle, glycerol injury induces expression of early adipogenic markers, such as PPAR gamma and CEBP alpha as soon as three days post-injury. Mature markers such as adiponectin and perilipin can be detected as early as five days after glycerol injury.
Using genetic lineage tracing, the majority of PDGF receptor alpha FAPs express the lineage marker EYFP. Seven days post glycerol injury, EYFP positive FAPs have turned into EYFP positive perilipin expressing adipocytes, demonstrating that FAPs are indeed the cellular origin of intramuscular fat. The protocol can also be adapted to visualize the myogenic compartment.
Using antibodies against PAX7 and MYOD1, muscles stem cell and myoblast markers, respectively can be readily detected five days post glycerol injury, even in PFA fixed muscle tissue sections. This protocol can also be adapted to visualize and quantify adipocytes and other adult tissues. In addition, our tissue preservation technique is compatible with the variety of different standing techniques.
