Cy3 streptavidin

Adult mice were anesthetized with an overdose of pentobarbital and then transcardially perfused with 4% paraformaldehyde (PFA). Mouse brain was dissected and fixed in 4% PFA for 4 h. After cryoprotection in 30% sucrose, brain sections (35 μm) were cut on a cryostat microtome (Leica CM1950, Leica Biosystems, Wetzlar, Germany). After rinsing with PBS and 0.3% Triton-X in 0.1 M PBS (PBST), the brain sections were blocked with 2% (w/v) bovine serum (BSA) in PBST for 1 h. Then, the brain sections were incubated with primary antibodies at 4°C for 48 h and secondary antibodies at room temperature for 2 h. Images were collected using a Zeiss LSM510 Meta or Nikon A1 confocal microscope and analyzed using FIJI. The antibodies used were as follows: anti-choline acetyltransferase (1:200, goat, AB144P, MERCK, Kenilworth, NJ, United States), anti-NK1R (S8305, rabbit, 1:5,000, Sigma-Aldrich, St. Louis, MO, United States), goat anti-rabbit for NK1R (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), donkey anti-goat for choline acetyltransferase (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), and Cy3-streptavidin (1:500).

DNA FISH was performed as follows. Cells grown on coverslips were fixed with 4% formaldehyde and 0.5% Triton X-100 in PBS for 15 min and then permeabilized with 0.5% saponin and 0.5% Triton X-100 in PBS for 20 min. Samples were immersed in 20% glycerol in PBS for 30 min and subjected to four cycles of freeze–thawing by freezing the cells in liquid nitrogen for 30 s each time and then thawing at room temperature. The cells were then treated with 0.1 N HCl for 15 min. For denaturation and hybridization, the cells were incubated in hybridization mixtures (2 × SSC, 50% formamide, 10% dextran sulfate, 1 mg/mL tRNA, and 5–10 μg/mL probe DNA) at 75 °C for 4–10 min (for denaturation of genomic DNA) and then at 37 °C for 48–72 h (for hybridization). For RNA FISH, the same protocol was used, except for the genomic DNA denaturation step. BAC probes were labeled with biotin or digoxigenin in a nick translation mixture (Roche), according to the manufacturer’s protocol. After hybridization, the cells were washed with 2 × SSC and 50% formamide at 37 °C for 5 min, followed by 2 × SSC at 37 °C for 5 min. FISH signals were detected with fluorescein isothiocyanate-anti-digoxigenin (Roche) or Cy3-streptavidin (Jackson ImmunoResearch). DNA was counterstained with 4,6-diamidino-2-phenylindole.