The medullary segment containing the TNC between +1 and −5 mm from the obex was removed, post-fixed for 24 h in the same fixative and subsequently transferred in solutions of sucrose at increasing concentrations (up to 30%) during the following 72 h.
The mouse brain was removed, post-fixed, rinsed, sliced on a vibrotome, and mounted on slides.
The brain was removed, post-fixed overnight at 4°C and cryosectioned.
The lumbar region of the spinal cord was removed, post-fixed in 4% PFA for 6 hours and cryoprotected in 30% sucrose for a minimum of 8 hours.
Sensitivity of these RNAs to a 5' monophosphate-specific exonuclease indicates that the RIG-I-activating 5' triphosphate group was removed post-transcriptionally by a viral function.
The tibialis anterior muscle was removed, post-fixed in 4% PFA overnight at 4°C, then cryopreserved in 30% sucrose at 4°C overnight.
The lumbar enlargement of the spinal cord was removed, post-fixed in 4% PFA overnight at 4°C, then cryopreserved in 30% sucrose at 4°C overnight.
The brainstem was removed, post-fixed in 4% paraformaldehyde for 7 days, and cut into 80-µm sections with a vibratome.
The lumbar spinal cord was removed, post-fixed in 4% PFA for 30 minutes at 4°C and osmotically dehydrated overnight in 30% sucrose at 4°C. 20 µm transverse sections were subsequently processed using a cryostat CM 300 (Leica) for immunostaining.
For the muscle preparation: the omohyoid muscle along with a short length of the innervating nerve was removed, post-fixed in 4% PFA for 30 min, rinsed in PBS (room temperature, 30 min×2), and then mounted on slides with the Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Then the brain was removed, post-fixed for 3 h, and cryoprotected for 24 h in PBS containing 20% sucrose.
