Cy3 conjugated donkey anti mouse

Skin biopsies (3 mm) were obtained, processed and analyzed according to consensus standards (Lauria et al., 2010 (link)). Tissue was placed in Zamboni's fixative (NewcommerSupply, Middleton, WI, USA) for 24hrs at room temperature, rinsed with 0.01M phosphate buffered saline (PBS) and placed in cryoprotectant (20% glycerol in 0.1M Sorrenson's phosphate buffer) for a minimum of 24hrs at 4°C. Serial frozen sections 70μm thick were made. Sections were permeabilized in 0.5% Triton X-100 for 30 minutes at room temperature, incubated in TNB (0.1M Tris HCl, 0.15M NaCl and 0.5% Boehringer milk powder) with 1% Triton X-100 for 2hrs at room temperature, then treated overnight at 4°C with primary mouse antibodies against myelin basic protein (Calbiochem Cat#NE1019-100ul, 1:1000) and anti-human PGP9.5 from rabbits (ABDserotec Cat#7863-0504, 1:1000) diluted in TNB with 0.5 % triton X-100. Sections were washed in 0.01M PBS with 0.5% triton X-100 and fluorescent secondary antibodies FITC-conjugated donkey anti-rabbit and Cy3-conjugated donkey anti-mouse (Jackson ImmunoResearch; 1:500) applied for 2hrs at room temperature. Sections were washed in 0.01M PBS with 0.5% Triton X-100, then 1mM CuSO₄ for 10 minutes, and mounted on glass slides with VECTASHIELD Mounting Medium (Vector Labs, Burlingame, CA).