Chicken anti nefh

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.

Skin was dissected from 8 week old adult mice and post-fixed in 4% PFA for 30 min at RT. DRG were cryo-protected overnight at 4°C in 30% sucrose-PBS, embedded in OCT, and then sectioned at 18 μm onto slides. Briefly, slides were blocked in 5% normal goat serum in PBST (0.1% Triton X-100) and incubated overnight at 4°C in 1:1000 primary antibody in blocking buffer. Slides were washed 3X in PBS then incubated 45 min at RT in 1:1000 secondary antibody. Slides were washed 5X in PBS and mounted in Fluoromount-G +DAPI with No. 1.5 coverglass. Primary antibodies used: Rabbit anti-DsRed (Clontech #632496), Chicken anti-NefH (Abcam #4680), Chicken anti-β-tubulin III (Abcam #107216), mouse anti-PGP9.5 (Abcam #8189). Secondary antibodies used: Goat anti-Mouse Alexa 488 (Abcam #150117), Goat anti-Rabbit Alexa 594 (Invitrogen #R37117), Goat anti-Chicken Alexa 488 (ThermoFisher #A11039). For co-localization analysis, only fibers for which >50% of the length of the visible fiber contained co-localized (white) pixels were counted. Image analysis was performed using FIJI.