Fig. 3. Localisation of vasoactive intestinal polypeptide (VIP)-binding sites to the chromaffin cell subtypes in the posterior cardinal vein of rainbow trout using triple labelling. (A) Dopamine ß-hydroxylase (DßH)-immunoreactive cells (mouse anti-DßH). (B) Phenylethanolamine N-methyl transferase (PNMT)-immunoreactive cells (rabbit anti-bovine PNMT). (C) Labelling of VIP-binding sites. (D) Overlay image of the sections shown in A and B (DßH- and PNMT-positive cells); the PNMT-negative cells are indicated by arrows. (E) Overlay image of the sections shown in A and C (DßH and VIP labelling). (F) Overlay image of the sections shown in B and C (PNMT and VIP labelling); the PNMT-negative cells are indicated by arrows. Yellow/orange colour indicates co-localisation. Note that only a few of the DßH-labelled cells were negative for PNMT, thus indicating only a small number of noradrenaline-containing cells. Moreover, the pattern of VIP binding was identical to that of DßH, indicating that all chromaffin cells possess VIP-binding sites. Scale bars A–F, 150 µm.