Goat anti phox2b

IF and cryosectioning were performed as previously described (Huang et al., 2012 (link)). Briefly, frozen sections were cut with 20 μm thickness. The primary antibodies and antiserum used are: chicken anti-GFP (1:1000, Abcam) and goat anti-Phox2b (1:500, Santa Cruz). Secondary antibodies were conjugated with Alexa Fluor 488 or 555 (1:2000, Molecular Probes). We used a Leica TCS SP5 confocal system to detect fluorescent staining. Image brightness and contrast were normalized using Image J and Adobe Photoshop.

Mice were perfused transcardially with 10 mL of ice-cold PBS followed by 10 mL of 4% ice-cold PFA. Brain and nodose jugular ganglion complex were dissected and post-fixed in 4% PFA at 4°C overnight and then transferred to 30% sucrose in PBS at 4°C for two days. Tissue was embedded in the optimum cutting temperature compound (OCT) and stored at −80°C until use. Tissues were sectioned at 16–30 microns using a Leica CM3050 cryostat and mounted onto Superfrost Plus microscope slides (Thermo Fisher Scientific).
For immunofluorescent staining, the sections were briefly washed by PBS with 0.1% Tween 20 (PBST), permeabilized with 0.3% PBS Triton X-100 for 30 mins, and then blocked by 2% bovine serum albumin (BSA) in PBS for 1 hr. Tissue sections were incubated with primary antibody overnight at 4 °C. The primary antibody used as: goat anti-Phox2b (Santa Cruz, sc-13224,1:150). After washing with PBST, secondary antibodies (donkey anti-goat, Invitrogen, A-21447, 1:500) were applied for 2 hours at room temperature. Then the samples were washed with PBST three times (5 min each time). Sections were stained with 4’,6 -diamidino-2-phenylindole, di-hydrochloride (DAPI, Invitrogen, D1306), before mounted prolong gold antifade reagent (Invitrogen, P36930). Fluorescent images were obtained on a Nikon A1 confocal microscope.