Sylgard medium

Animals were euthanized with ketamine (300 mg/kg), xylazine (15 mg/kg) and acepromazine (6 mg/kg) 5 weeks following SCI. The hemi-diaphragm was exposed laterally along the rib cage and then excised using spring scissors (Fine Science Tools, Foster City, CA), stretched flat, pinned down to Sylgard medium (Fisher Scientific, Pittsburgh, PA), and washed with PBS. Diaphragm was then fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min, followed by a wash in PBS. Superficial fascia were then carefully removed from the surface of the diaphragm, and diaphragm muscles were processed for NMJ immunohistochemistry. After hemi-diaphragm was removed, animals were perfused with 0.9% saline solution, followed by 4% paraformaldehyde. Spinal cord and brain were dissected and post-fixed in 4% paraformaldehyde overnight at 4°C. The tissue was then washed with 0.1M phosphate buffer solution for 24 hours and cryoprotected with a 30% sucrose solution for 3 days. The cervical spinal cord was embedded in tissue freezing medium, flash frozen, and sectioned in transverse or sagittal orientations at 20 μm thickness.

Fresh hemi-diaphragm muscle was dissected from each animal for whole-mount immunohistochemistry, as described previously (Wright et al., 2007 (link)). Hemi-diaphragm muscle was dissected, stretched, pinned down to Sylgard medium (Fisher Scientific, Pittsburgh, PA), and extensively cleaned to remove any connective tissue to allow for antibody penetration. Motor axons and their terminals were labeled with SMI-312R (Covance, Princeton, NJ; 1:1000) and SV2-s (DSHB, Iowa City, IA; 1:10), respectively, and both primary antibodies were detected with FITC anti-mouse IgG secondary (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:100). Post-synaptic acetylcholine receptors were labeled with rhodamine-conjugated alpha-bungarotoxin (Life Technologies, Grand Island, NY; 1:400). Labeled muscles were analyzed for total numbers of NMJs and intact, denervated and multiply-innervated NMJs. Whole-mounted diaphragms were imaged on a FluoView FV1000 confocal microscope (Olympus, Center Valley, PA). We only conducted NMJ analysis in ipsilateral hemi-diaphragm because in our previously published work we did not observe denervation or sprouting in contralateral hemi-diaphragm after cervical hemi-contusion SCI (Nicaise et al., 2012 (link)).

Fresh hemi-diaphragm muscle was dissected from each animal for whole-mount immunohistochemistry, as described previously (Nicaise, Hala, Frank, et al., 2012a (link)). The muscle was dissected, stretched, pinned down to Sylgard medium (Fisher Scientific) and cleaned. Motor axons and their terminals were labeled with SMI-312R (Covance, Princeton, NJ; 1:1000) and SV2-s (DSHB, Iowa City, IA; 1:10), respectively. Detection of these antibodies was made with FITC anti-mouse IgG secondary (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:100). Postsynaptic acetylcholine receptors were labeled with rhodamine-conjugated alpha-bungarotoxin (Life Technologies; 1:400). Labeled muscles were imaged using a FluoView FV1000 confocal microscope (Olympus, Center Valley, PA) and analyzed for total numbers of NMJs and intact and denervated NMJs.