Immunoelectron microscopic visualization of neurohypophyseal hormones: evaluation of some tissue preparations and staining procedures.

To obtain optimal electron microscopical localization of vasopressin in the rat neurohypophyses two immunocytochemical staining procedures and several tissue treatments were evaluated. The peroxidase-antiperoxidase technique allowed greater dilution of the first antibody than the indirect method using a commercial peroxidase conjugate. This appeared crucial for the dilution-dependent specificity in the localization of vasopressin. Immersion fixation with formalin gave better results than those obtained with perfusion fixation with glutaraldehyde-paraformaldehyde (resulting in similar preservation of immunoreactivity) and freeze substitution (showing the best preservation of immunoreactivity). However, these latter two tissue fixation methods resulted in less than optimal preservation of general ultrastructure than immersion fixation in formalin alone. Immersion fixation with glutaraldehyde-paraformaldehyde followed by OsO4 improved ultrastructural detail, but immunoreactivity decreased considerably. Fixation with paraformaldehyde-picric acid resulted in poorest preservation of morphologic detail. Immunoreactivity was similar in both Epon 812 and Araldite 6005 embedded tissue.