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Immunofluorescent staining using the Neuropeptide S antibody (ab18252) in the rat hypothalamus. Picture taken using objectives X20 (A) or X40 (B). The staining is observed in thin and varicose fibers. This staining is blocked by preincubation with immunising peptides (ab18698 and ab18699). ab18252 was used at 1/1000 (0.3µg/ml) incubated overnight at room temperature. Secondary antibody used was anti-rabbit Alexa Fluor 488 at 1/1000 incubated for 2 hours at room temperature. Rat brain tissue was perfusion fixed with 4% PFA followed by overnight post-fixation in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut on a cyrostat and immunohistochemistry performed by the 'free floating' technique.

IC50: 46 fmoles; ab18252 used at a dilution of 1/3000; Tracer: [125I] Y10-hNPS, 12.000 cpm; Detection method: anti-rabbit SPA (Amersham).

Radioimmunoassay was performed in equilibrium, using the SPA technology (Amersham). 50µl antirabbit YSi SPA beads (RPN 140) were mixed with 50µl antiserum (1:3000), 50µl tracer (12,000 cpm) and with or without cold NPS peptide in increasing concentrations. All reagents were resuspended in 0.1M phosphate buffer, pH 7, 0.1% BSA. Incubation was for 48h at RT. Bound radioligand was detected by scintillation proximity in a beta counter (MicroBeta, Wallac).