Uchl1 pgp9.5 polyclonal antibody

Immunohistochemistry was performed on a fourth separate cohort of male and female mice (8 weeks on DOX) as previously described (Toma et al., 2017 (link)) (n = 8/sex/genotype). Briefly, mice were overdosed with vaporized isoflurane and then perfused transcardially with saline followed by 4% paraformaldehyde. The glabrous skin of the hind paw was excised, placed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4), and stored overnight at 4 °C. The samples were embedded in paraffin, 25-μm-thick sections cut, deparaffinized, and incubated with a UCHL1/PGP9.5 polyclonal antibody (Proteintech, Cat. # 14730-1-AP; Rosemont, IL, USA) and goat anti-rabbit IgG (heavy and light chains) secondary antibody conjugated with Alexa Fluor 594 (A11037; Life Technologies, Eugene, OR, USA). Sections were examined using a Zeiss Axio Imager Z2 equipped with a motorized XY stage, microcator and MicroBrightfield Neurolucida and Stereo Investigator image analysis software (MBF Bioscience, VT, USA). Sections were examined in a blinded fashion under 63× magnification. Main fibers were counted in real time by dynamically focusing and following nerve fibers once they crossed the junction of the basement membrane. Density of fibers is expressed as fibers/mm.