Alexa fluor 488 conjugated to streptavidin

After resectioning, 50 µm sections were washed in PBS for at least 10 min prior to receiving 0.1M glycine made in 0.05M PBS for 30 min. The tissue was subsequently washed three times in PBS for 5 min each wash. Next, sections received 0.5% sodium borohydride in PBS for 15 min. Sections were then washed twice for 5 min in PBS before blocking in 5% NGS, 0.5% Tx, and 1% H₂O₂ in PBS for 30 min. After blocking, sections received one of three primary antisera for two days: a monoclonal anti‐peripherin (1:300; Chemicon International, Temecula, CA), a polyclonal anti‐VIP (1:8,000; Immunostar, Inc. Hudson, WI), or a polyclonal anti‐MUC2 (3 µg/ml; Novus Biologicals). After primary sections were washed with 1% NGS in PBS four times for 15 min each wash. Next, secondary antibody was added for 2 hr at room temperature and consisted of 1% NGS and 0.5% Tx in PBS with a biotinylated goat anti‐rabbit secondary antibody (1:2,500; Jackson Immunoresearch Inc. West Grove, PA). Secondary antibody was washed out with four 15 min washes composed of 0.02% Tx in PBS. Sections were next incubated with an Alexa Fluor 488 conjugated to streptavidin (1:500; Invitrogen) in 0.32% Tx in PBS for 1 hr. Finally, sections received three PBS washes prior to mounting and imaging.

Cultures were fixed in 4% formaldehyde—4% sucrose in PBS at 4°C for 24 hours. Cultures were then sliced at a thickness of 30 μm using a Vibratome V1200 (Leica Biosystems, Wetzlar, Germany) and mounted on Fisherbrand™ Superfrost™ microscope slides. Mounted sections were permeabilized in 0.15% Tween-20/0.05% Triton (Sigma-Aldrich, MO, USA) for 15 minutes and blocked in 10% normal goat serum (NGS) (Sigma-Aldrich, MO, USA) for 30 minutes, followed by blocking for endogenous avidin and biotin (avidin/biotin blocking kit, Invitrogen, MA, USA) per manufacturer’s instructions. Sections were then incubated with the primary antibodies mouse anti-TH (1:1000 EMD Millipore, MA, USA) and rabbit anti-SK3 (1:1,500 Alomone labs, Jerusalem, Israel) for 20 hours at 4°C followed by incubation with biotinylated goat anti-rabbit IgG (H+L) (Abcam, Cambridge, UK) for 15 minutes. Sections were incubated with the secondary antibodies goat anti-mouse Alexa Fluor® 568 (1:500) and Alexa Fluor® 488 conjugated to streptavidin (1:500) (Invitrogen, MA, USA) for 2 hours at room temperature. Sections were rinsed three times with PBS and cover-slipped using Prolong® Gold antifade reagent with DAPI (Invitrogen, MA, USA). All antibody solutions were made using 1% NGS/0.02% each Tween-20 and Triton in PBS.