C fos

To determine whether irritant exposure, restraint, or periorbital stimulation induced c-Fos expression in spinal trigeminal nucleus caudalis (TNC) neurons, immunocytochemistry was performed as described previously (19). Following overnight fixation, the tissue was rinsed in PBS and then cryoprotected in 10% sucrose for 2 hours followed by 20% sucrose in PBS overnight at 4°C. Frozen coronal sections (40 µm) of the brainstem and spinal cord were obtained serially through the medulla to the third cervical segment and collected free-floating in PBS. The obex was used as the most rostral point and was designated 0.0 mm. The first six serial sections from regions at 1.5, 3.0, 4.5 and 6.0 mm caudal to the obex were processed and examined in each animal. Sections were blocked in 4% normal goat serum and 0.025% Triton-X100 in PBS for 1 hour and incubated with the primary antibody (c-Fos; Santa Cruz (#sc-52); 1:2000) diluted in blocking solution overnight at 4°C. Subsequently, sections were rinsed in PBS, incubated with the secondary antibody (goat anti-rabbit IgG conjugated to HRP, Jackson ImmunoResearch; 4 µg/ml) for 1 hour at room temperature and processed for visualization using DAB. Cells were considered positive for c-Fos immunoreactivity (c-Fos-IR) if the nucleus was densely stained. No reactivity was observed in control sections in which primary antibody was omitted.

MLO-Y4, OCY454, and primary osteocytes were wounded by glass beads, as previously described (8 (link), 19 (link)), to facilitate wounding large numbers of cells. Vitamin E (Sigma T3251, 220 μM) (19 (link)) or calpeptin (Sigma C8999, 20 μM) (20 (link)) were introduced as indicated 24 hours prior to wounding. Whole cell lysates were collected for western blotting as previously described (21 ), using antibodies against c-fos (Santa Cruz) and β-actin (Sigma). Please see Supplementary Methods for additional details.
To assess cell survival after mechanical wounding, MLO-Y4 cells were wounded by glass beads in the presence of lysine-fixable fluorescein-conjugated dextran (10 kDa, 5 mg/mL+10 mg/mL BSA) containing either 1.8 mM Ca²⁺, 1.8 mM Ca²⁺+20 μM calpeptin, or 1.5 mM EGTA. Five minutes after wounding, cells were stained with propidium iodide (0.3 μg/mL) to detect dead cells (i.e., unrepaired PMD) and imaged on a multi-photon confocal microscope (Zeiss). Please see Supplementary Methods for additional details. The percentages of PMD-affected cells and dead cells were quantified (Bioquant).