*4.4. Immunohistochemistry and Peptide Absorption Studies*

Rats were anesthetized by intraperitoneal (i.p.) injections of ketamine (85 mg/kg) and xylazine (10 mg/kg). The anesthetized animals were perfused transcardially with 4% paraformaldehyde. The brainstem was harvested and stored in the phosphate buffer saline (PBS, pH7.4) containing 30% sucrose for at least 24 h. Then the tissue was frozen in powdered dry ice and stored at −80 ◦C. Serial horizontal sections were cut at 20 μm using a Leica CM1850 cryostat at −18 ◦C. Selected sections were thawed and mounted onto Superfrost/Plus slides. The sections were rinsed in PBS and then blocked in 10% normal donkey serum in PBS for 60 min. The sections were incubated with rabbit anti-TRPA1 antibody (1:100, Santa Cruz, Dallas, TX, USA) overnight at 4 ◦C and FITC donkey anti-rabbit IgG (1:100, Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA). Images were captured by a fluorescence microscope.
