*4.7. Immunofluorescence*

Animals were sacrificed after the behavioral testing on days 3 and 14. Rats were deeply anesthetized using pentobarbital (80 mg/kg, i.p.) and transcardially perfused with 150 mL normal saline (4◦C) and 400 mL 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for prefixation. The L4–6 segments of the dorsal root ganglion (DRG) and lumbar spinal cord were removed and postfixed in 4% paraformaldehyde for 3 hours at 4 ◦C before transfer to 15% and 30% sucrose for dehydration. Tissues were embedded in Tissue-Tek O.C.T compound (SAKURA, Torrance, CA, USA). DRGs were cut at thickness of 14 μm, and frozen sections of the lumbar spinal cord were cut at thickness of 20 μm using a CryoStar (NX50 HOP, Thermo, Walldorf, Germany).

Sections were rinsed with TBST (0.1 % Tween-20) and blocked with 5% normal donkey serum for one hour at 37 ◦C. Sections were then incubated with rabbit anti-P2X3 (1:800 in 5 % normal donkey serum, Alomone, Jerusalem, Israel) overnight at 4 ◦C. The slides were then incubated in Alexa Fluor 647-conjugated AffiniPure donkey anti-rabbit IgG (H + L) (Jackson, West Grove, PA, USA) for one hour at 37 ◦C. Images were taken using the A1R confocal microscope (Nikon, Tokyo, Japan). We used NIS-Elements AR to calculate the mean intensity of P2X3-immunoreactivity (-ir) in the region-of-interest (ROI) in DRG and SCDH. The relative level of P2X3 in each group was normalized to the expression in the control group.
