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. 2022 May 5;133(1):41–59. doi: 10.1152/japplphysiol.00088.2022

Table 4.

Example protocol of processing transfected skeletal muscles for immunohistochemistry

  1. Muscle Pinning Step: After harvest, TA muscles are pinned on cork at the insertion and origin ends of the muscle and placed in 5-mL tubes containing 4% paraformaldehyde (PFA) solution. The 5-mL tubes can be kept on ice until all TAs have been harvested. Once all the TA muscles have been collected, the 5-mL tubes are placed at 4°C for at least 16 h.

  2. Optional: A third pin can be used in the middle of the TA at the mid belly. Using a razor blade, the TA muscle is cut in half with the TA origin portion (upper half) being used for histology. The TA Muscle remains on the cork and pinned all the way through to step 3. The other half of the TA muscle can be frozen in liquid nitrogen and stored at −80°C until a later date for biochemical analyses.

  1. Sucrose gradient step: The next morning, remove the 4% PFA and rinse the muscles twice with chilled 1× PBS (5 mL each time). Then place the muscles in 10% (wt/vol) sucrose solution (made in 1× PBS) at 4°C for 2–3 h. Following the 2- to 3-h incubation, switch to 20% sucrose solution for 2–3 h at 4°C. Finally, switch the muscles to 30% sucrose for 16 h or overnight at 4°C. (This is all performed in the original 5-mL tube).

  2. Following incubation in 30% sucrose, embed muscle in freezing medium (optimal cutting temperature; OCT). Remove pins and orientate the TA muscle in the OCT-filled cryomold (use a sharpie on sides to mark the size and location of the muscle in the cryomold). By marking the cryomold for the muscle orientation and location it will help with the sectioning phase. Slowly lower the cryomold into a small polystyrene box of liquid nitrogen to freeze the OCT using forceps until the OCT has frozen around the TA muscle. Never fully submerge the mold into the liquid nitrogen.

  3. The cryomold is now ready to be used on the cryostat for sectioning at 10 µm size. Sections can be mounted, cover slipped, and visualized under the fluorescent microscope using appropriate color channel.

TA, tibialis anterior.