Anti collagen 4 antibody

For quantification of IENFs, 3 mm² biopsies from the plantar surface of the hind paws were collected 3 weeks after completion of paclitaxel treatment) and processed as described [4 (link); 5 (link); 34 ; 38 (link)]. Biopsies were immediately placed in Zamboni’s fixative for 24 hrs., transferred to 20% sucrose, frozen in Optimal Cutting Temperature compound (OCT), and sliced into 25 μm sections. The sections were blocked for 2 hrs. At room temperature in 0.1 M PBS containing 5% normal donkey serum/0.3% Triton X-100. Sections were incubated with an antibody against the pan neuronal marker PGP9.5 (AbD Serotec; Rabbit, 1:1000) along with anti-Collagen IV antibody (Southern Biotech; Goat, 1:100) followed by Alexa-594-donkey anti-rabbit (Life Technologies, 1:500) and Alexa-488-donkey anti-goat (Invitrogen, 1:500). Secondary antibodies alone did not give a signal, indicating specificity of the staining. Three randomly chosen sections from each paw were quantified under a Leica fluorescence microscope. Nerve fibers that crossed the collagen stained dermal/epidermal junction into the epidermis were counted in three fields of each slice using the 40× objective. The length of the epidermis within each field was measured using ImageJ. IENFs density was determined as the total number of fibers/length of epidermis (IENFs/mm). A scorer blinded to the experimental set up rated the IENF density.