Mouse Tissue Collection

For RT-PCR and RT-qPCR, mice were euthanized via CO₂ overdose and tissues were dissected and stored in ice cold RNeasy Kit Lysis Buffer (Qiagen, Germany). Samples were processed for RNA extraction immediately after dissection.

For histopathology and LacZ staining, mice were transcardially perfused with ice cold phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) for fixation. Dissected tissues were fixed on ice as follows: skin in 1% PFA 1 hour, DRG and TG in 2% PFA 30 minutes, spinal cord (SC) in 2% PFA 1 hour, and brain in 2% PFA 2 hours. After fixation, tissues were immersed in 30% sucrose for 24 hours at 4°C and embedded in OCT (Sakura, Torrance, CA) for frozen sectioning. Mouse DRGs and SCs used for immunostaining were fixed in 4% PFA on ice for 90 minutes before sucrose incubation and frozen sectioning. For flow cytometry, skin was harvested without fixation and processed immediately.

For ELISA, mouse serum was isolated from blood collected by orbital bleed by centrifugation at 10,000g for 10 minutes at 4°C on experimental day 6. ELISAs for IgE (Biolegend 432404, San Diego, CA) and TARC/CCL17 (R&D DY529–05, Minneapolis, MN) were performed according to the manufacturers’ instructions.