Mouse anti calcitonin gene related peptide

Mice were transcardially perfused with 50–100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) after they were deeply anesthetized with overdose of isoflurane. The L3/4 DRGs were collected, post-fixed with same perfusion solution for 2–4 hours and cryoprotected in 30% sucrose in 0.1 M PB for 48 hours at 4°C. The tissues were cut on a cryostat at the thickness of 15 μm. All sections were pretreated with acetone for 25 minutes and incubated at 37°C for 1 hour in 0.01 M phosphate buffer saline containing 0.3% Triton X-100 and 5% normal goat serum. The sections were then incubated at 4°C overnight with primary rabbit anti-ELF1 (1:400, biorbyt) alone or with a combination of primary rabbit anti-ELF1 and biotinylated isolectin B4 (IB4, 1:100; Sigma), mouse anti-neurofilament 200 (NF200; 1:500, Sigma-Aldrich, St. Louis, MO), mouse anti-NeuN (1:50; Gene Tex, Irvine, CA), mouse anti-calcitonin gene-related peptide (CGRP, 1:50; Abcam) or mouse anti-glutamine synthetase (GS, 1:500; EMD Millipore, Burlington, MA). Appropriate conjugated secondary antibodies were used. Under a Leica DMI4000 fluorescence microscope (Leica) with DFC365 FX camera (Leica), the immunofluorescent images were captured.