The use of peroxidase substrate Vector VIP in electron microscopic single and double antigen localization

Very few chromogens used in immunoperoxidase reactions can be combined to simultaneously localize two neural antigens with different labels at both light (LM) and electron (EM) microscopic levels. The objective of this study was to investigate the EM properties of a novel purple chromogen introduced by LM immunostaining by Vector Laboratories under the commercial name Vector VIP. The Vector VIP (VIP) was employed to demonstrate anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), retrogradely transported cholera toxin subunit B (CTB) and acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in single and double antigen immunostaining in combination with the chromogen 3,3'diaminobenzidine (DAB). The VIP reaction product proved resistant to loss during post-fixation in OSO4 and dehydration in acetone. In EM preparation, the VIP reaction product was granular in appearance and easily distinguishable from the diffuse reaction product of DAB. Compared to the chromogen benzidine dihydrochloride (BDHC), the VIP reaction procedure is much simpler, more sensitive and consistently generates the same texture of the electron-dense precipitate. This study demonstrates the usefulness of VIP as a chromogen for correlative LM and EM immunoperoxidase staining. The VIP can be used either in single or double immunostaining in combination with DAB. In addition, we have examined the EM properties of another commercial chromogen, peroxidase substrate Vector SG (SG). The blue-gray reaction product of this chromogen is strongly osmiophilic and the electron-dense precipitate appears amorphous.