N9528

The 48-h fasted zebrafish were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Brain sections (14 µm) of the diencephalon were treated with 3% H₂O₂ in PBS for 15 min, and 1% BSA and 0.2% Triton X-100 in PBS for 30 min. And then, the sections were incubated with a primary antibody against NPY (N9528, Sigma-Aldrich, St Louis, MO, 1:5000) overnight at room temperature, with biotinylated anti-goat IgG solution (BA1000, Vector Labs, Burlington, CA, 1:200) for 3 h at room temperature, and with Vectastain ABC reagents (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The sections were visualized with 0.02% diaminobenzidine tetrahydrochloride and 0.005% H₂O₂ in 0.05 M Tris-HCl buffer for 20 min and observed using a light microscope (BX51, Olympus Optical Co. Ltd., Tokyo, Japan). Nissl bodies were counterstained with 0.5% cresyl violet solution.

Immunohistological assessment was also made for NPY, as described by Schwarzer et al,. 1996 (link) and for parvalbumin, as described by Yew et al,. 1990 (link). After suppressing the endogenous peroxidase activity, the free-floating sections were incubated with the primary antibody (rabbit anti-NPY, 1:3,000, Sigma N9528, for 48 h; mouse anti-PARV, 1:1,000, Sigma P3088, for 24 h) in a solution containing blocking buffer (3% bovine fetal serum, 0.2% Triton X-100 in PB). Immunostaining was visualized using a biotinylated secondary antibody for 2 h (1:200, Vector), the avidin-biotin complex and revealed with diaminobenzidine tetrahydrochloride (DAB, 1 mg/1 ml). Cell countings were performed on three consecutive slices per animal using a ×40 objective with ×10 digital zoom (×400 magnification, Olympus B×50). On each slice, a microscope grid was placed over three different sample areas (1000 μm²) per region, which included the pyramidal layer of CA1/CA3, granular layer of dentate gyrus, hilar region, basolateral and basomedial amygdala, entorhinal and piriform cortex, from rostral to dorsal levels (between Plates 26 and 42; Swanson, 2004 ). The result attributed to each animal was an average value, from both hemispheres, and described as the total number of neurons per mm².

For immunocytochemistry (ICC) staining, neurons were fixed in 2% PFA for 15 min at room temperature. The coverslips were washed three times with 7.5 mM glycine (Sigma-Aldrich, Germany) in 1xPBS to block unreacted aldehydes following a single wash in 1xPBS and stored in 1xPBS at 4°C until ICC was performed. Coverslips were transferred to a humidifying chamber and washed three times with 1xPBS before being incubated in blocking buffer containing 5% goat serum, and 0.25% Triton X-100 for 30–45 min. The coverslips were then incubated with rabbit anti-NPY antibodies recognizing the C-terminal region of full-length NPY (1:1000; N9528, Sigma-Aldrich, Germany) and chicken anti-MAP2 (1:500, Ab5392, Abcam) in 5% goat serum + 0.25% Triton X-100 (Sigma-Aldrich, Germany, #93443) at 4°C overnight. The following day, the coverslips were washed four times with 1xPBS for 5 min before incubating with the secondary antibodies (1:500; goat anti-rabbit, Alexa 568 and goat-anti chicken, Alexa 488; Invitrogen) for 1 h at room temperature in 5% goat serum + 0.25% Triton X-100 excluded from light exposure. Next, the coverslips were washed three times with 1xPBS for 5 min, mounted on slides with DAPI Fluoromount-G (SouthernBiotech, #0100-20), and allowed to dry overnight. The stained coverslips were stored at 4°C excluded from light exposure.