*2.9. Western Blot*

Brain samples of young and old fish were extracted in RIPA buffer (Radio Immuno Precipitation Assay) with Lysis buffer (50 mM Tris–HCl pH 7.4, 1% Triton X-100, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA), containing protease inhibitors 2 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA). Samples were homogenized with an Ultra-Turrax T25 (IKA Labortechnik, Staufer, Germany) at 13,500 rpm. Homogenates were centrifuged 15,000 rpm for 30 min at 4 ◦C; supernatants were collected separately and the protein concentration was determined with Bio-Rad dye protein assay (Bio-Rad Laboratories Inc., Hemel Hempstead, UK). For each sample, 20 μL of protein were boiled at 100 ◦C for 10 min in 20 μL of 2× loading buffer (50 mM Tris–HCl pH 6.8, 100 mM b-mercaptoethanol, 4% SDS, 0.1% blue bromophenol, 10% glycerol). Proteins were separated on a 12% SDS–polyacrylamide gel electrophoresis with 4% stacking gel in 1% Tris–glycine buffer (0.025 M Tris, 0.190 M glycine, and 0.1%SDS [pH 8.3]) in a miniprotean cell (Bio-Rad) at 100 V for 2 h. The separated proteins were electro-transferred on a nitrocellulose membrane with transfer buffer (48 mM Tris base, 39 mM glycine, 0.04% SDS, and 20% methanol [pH 8.5]) in a minitransfer cell (Bio-Rad) at 100 V at 4 ◦C for 1 h. Membranes were incubated at 4 ◦C for 1 h in blocking buffer containing 1% PBS and 0.05% Tween 20 with 5% dry non-fat milk and probed with polyclonal antibody raised in rabbit against Nesf-1 (H-003-24; Phoenix Pharmaceuticals; Belmont, CA, USA) and β-actin (A5060, Sigma, St. Louis, MO, USA) used as an internal marker. Primary antibody was diluted 1:2000 and incubated overnight at 4 ◦C, followed by incubation with the secondary goat anti-rabbit IgG (Sigma 1:3000) antibody for 1 h at RT. Signals were detected by chemoluminescence with the Pico Enhanced Chemiluminescence Kit (Pierce Chemical) with Chemidoc (Bio-Rad, Hercules, CA, USA). A pre-stained molecular-weight ladder (Novex Sharp Pre-Stained Protein Standard, Life Technologies, Monza, Italy) was used to determine protein size. Specificity was determined by pre-absorption of primary antibodies with their relative control peptides before western blotting.

#### *2.10. Immunohistochemistry*

Immunohistochemistry (IHC) was conducted on both paraffin and cryosections of brain and intestine. Paraffin slides were deparaffinized in xylene and rehydrated in progressively diluted alcohols, while cryosections were dried for 2 h at RT, placed in a bath of acetone 100% for 10 min at 4 ◦C, air-dried for a few minutes, and washed once in water and twice in 1× PBS. Then, both slides were treated for 30 min with 3% H2O2 and, after washing with 1× PBS, were incubated in normal goat serum (1:5 in 1×

PBS) at RT for 30 min. Incubation with primary polyclonal antibody raised in rabbit against Nesf-1 (1:1000, H-003-24; Phoenix Pharmaceuticals; Belmont, CA, USA) was performed at 4 ◦C ON. Sections were rinsed in 1× PBS for 15 min and incubated with EnVision reagent (DAKO, K406511) for 30 min at RT. Immunoreactive sites were visualized using a fresh solution of 10 μg of 3,3- -diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, #D5905) in 15 mL of a 0.5 M Tris buffer.
