Rabbit polyclonal anti trpa1 antibody

Total proteins of DRG were electrophoretically separated on 4–15% gradient SDS–PAGE gels and transferred onto nitrocellulose membranes. Membranes were probed with the following primary antibodies: mouse monoclonal anti-ZBTB20 antibody 9A10 (1:1,000); rabbit polyclonal anti-TRPA1 antibody (1:200, Abcam); rabbit polyclonal anti-TRPM8 (1:200, Abcam); goat polyclonal anti-TRPV1 (1:200, Santa Cruz Biotechnology); mouse monoclonal anti-α-tubulin antibody (1:8,000, Sigma-Aldrich). Secondary antibodies were either horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:8,000, Vector Laboratories). Signals were then generated using a Chemiluminescent Detection Kit (ECL Plus; Amersham Pharmacia Biotech) and visualized with a scanner. Six different preparations of control and mutant DRG individually were used for western blot analysis. Expression levels of each protein of interest were normalized to that of α-tubulin.

For immunostaining on frozen sections, the embryos were collected in ice-cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS and saturated with 20% sucrose in PBS overnight at 4 °C. For adult mice, after perfusing with 4% paraformaldehyde in PBS, DRG were dissected and collected in 4% paraformaldehyde for 1 h and saturated with 20% sucrose in PBS overnight at 4 °C (ref. 31 (link)). Cryostat sections of L4-5 DRGs were processed for single- or double- immunofluorescent staining with mouse monoclonal anti-ZBTB20 antibody 9A10 (1:1,000), goat polyclonal anti-peripherin antibody (1:200, Santa Cruz Biotechnology CA, USA), rabbit anti-neurofilament (N200) antibody (1:1,000, Sigma-Aldrich, MO, USA), lectin IB4-biotin (1:1,000, Sigma-Aldrich), goat polyclonal anti-CGRP antibody (1:4,000, Abcam, MA, USA), rabbit anti-Na_v1.8 antibody (1:1,000; Alomone Labs, Jerusalem, Israel),goat polyclonal anti-TRPV1 antibody (1:200, Santa Cruz Biotechnology), rabbit polyclonal anti-TRPA1 antibody (1:2,000, Abcam) and rabbit polyclonal anti-TRPM8 antibody (1:2,000, Abcam).