Quantifying Macrophage and Schwann Cell Recruitment in Peripheral Nerve Regeneration

S100β eGFP mice were euthanized at 10 days after repair (for both treatment groups) to determine macrophage recruitment and SC migration. The regenerative bridge was harvested within the conduit by transecting the proximal and distal sciatic nerve stumps 1 mm from the end of the 5-mm conduit. The epineurial sutures were cut and the regenerative bridge was removed from the conduit and fixed overnight in 4% paraformaldehyde. To stain macrophages, the bridge was then incubated in goat-blocking serum and casein mixture for 5 h. The sample was washed in PBS and then incubated in macrophage-specific primary antibody (1:50 CD68, Abcam) overnight. After the wash step, the regenerative bridge was transferred into secondary antibody solution (1:200 biotinylated goat anti-rabbit IgG, Vector Laboratories) and incubated overnight. Sample was washed again in PBS and transferred into Texas-Red conjugate solution (1:200, Life Technologies) and incubated overnight. The sample was then washed and incubated in DAPI solution (1:1000) overnight. After the final wash in PBS, the sample was immersed into DIX solution (a mixture of n-methyl-d-glucamine, diatrizoic acid, 60% iodixanol and 2% sodium azide) to clear the tissue. All the wash steps were done twice in PBS over a rocking table for 30 min each. All the incubation steps were done at room temperature on a turning wheel at 20–40 rpm.
Samples were then mounted on slides while immersed in DIX solution and the entire regenerative bridge was imaged using tiled, Z stack imaging (Zeiss 710 confocal). The macrophage and SC presence and distribution within the regenerative bridge was quantified by the summation of the fluorescent intensity of each channel (GFP green: SC, Texas red: macrophage, DAPI blue: all cells) at 250 µm intervals from the distal end of the proximal stump using ImageJ. Results were analyzed with group—agarose only (control) or agarose with IL10 (15 µg/mL) concealed to the observer. Differences in total numbers of macrophages were determined using two-tailed t test with P < 0.05.