Staining was performed according to Marshall et al. (2016) (link). Briefly, 8-week-old mice were euthanized and the back skin was shaved, depilated, and tape-stripped. The removed skin was fixed overnight in 4% PFA, then washed in PBS (3X for 10 min each). Dermal fat was scraped away with a scalpel and skin was washed in PBST (0.3% Triton X-100; 3X for two hours each) then incubated in 1:500 primary antibody (Rabbit anti DsRed: Clontech #632496; Chicken anti-Nefh: Abcam #4680) in blocking buffer (PBST with 5% goat serum and 20% DMSO) for 5.5 days at 4°C. Skin was washed as before and incubated in 1:500 secondary antibody (Goat anti-Rabbit Alexa 594; Invitrogen #R37117; Goat anti-Chicken Alexa 488; ThermoFisher #A11039) in blocking buffer for 3 days at 4°C. Skin was washed in PBST, serially dried in methanol: PBS solutions, incubated overnight in 100% methanol, and finally cleared with a 1:2 solution of benzyl alcohol: benzyl benzoate (BABB; Sigma) before mounting between No. 1.5 coverglass. Sectioned and whole mount skin samples were imaged on a Zeiss LSM 880 confocal microscope with OPO using a 20x water objective. Image analysis was performed using FIJI.
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