*2.8. In Situ Hybridization*

In situ hybridization (ISH) experiments have been conducted on brain and intestine sections and by means of sterile solutions and materials. Dyethilpyrocarbonate (DEPC) was added to millipore water (1 mL/L) to inactivate RNases, shaken and autoclaved. All solutions that were used in the steps until probe revelation were made with RNase free water and RNAse free conditions were kept during all prehybridization and hybridization steps.

Sections were dried for 2 h at room temperature (RT), washed well in 1× DEPC/PBS, and treated with 10 μg/μL Proteinase K (Sigma–Aldrich) 1:200 in 1× DEPC/PBS for 10 min. Proteinase K was inactivated by two washes in 2 mg/mL glycine, 5 min each. Sections were post fixed in 4% PFA for 20 min and well washed in 1× DEPC/PBS at RT. Prehybridization was carried out in a hybridization solution containing 50% formamide, 0.5% SSC (Saline-Sodium Citrate), 500 μg. Heparin, 50 μg/mL yeast RNA, and 0.1% Tween 20 at 55 ◦C for 1 h. Probes were denatured for 10 min at −80 ◦C and sections were then incubated ON at 55 ◦C in hybridization solution containing antisense NUCB2B probe at a concentration of 2 ng/μL. Post hybridization washes were carried out at 55 ◦C as follows: 2 × 20 min in 1× SSC, 2 × 10 min in 0.5× SSC and then in PBS added with tween20 (PBT) at RT. The sections were blocked with a blocking solution (BS) containing 10% normal sheep serum heat inactivated and 0.5% blocking reagent (Roche, Basel, Switzerland, cat. 11096176001) for 1 h at RT. Sections were incubated ON at 4 ◦C in fresh blocking buffer containing a 1:2000 dilution of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Roche, cat. 11093274910). After washing in PBS and levamisole (Vector Labs., Burlingame, CA, USA, SP-5000), the reaction was developed using Fast Red substrate (Roche, cat. 11496549001) under periodic inspection with an epifluorescence microscope. To stop the reaction, the sections were washed repeatedly in PBT and mounted with Fluoroshield Mounting Medium with DAPI (IBSC, Hermon, ME, USA, cat. AR-6501-0) as counterstaining for the nuclei.
