A triple ultrastructural immunogold staining method. Application to the simultaneous demonstration of three hypophyseal hormones.

The triple ultrastructural immunocytochemical labeling technique described here is based on the use of three different antisera raised in two different animal species: rabbit anti-corticotropin (R-ACTH), guinea pig anti-prolactin (GP-LTH) and rabbit anti-gonadotropin (R-LH beta). Staining is carried out on both sides (A and B) of the same tissue section. First, side A is incubated with a mixture of R-ACTH and GP-LTH and then with a mixture of the two corresponding species-specific immunoglobulins (IgG) adsorbed respectively to 5 and 20 nm gold particles: goat (G) anti-R IgG 5 + G anti-GP IgG 20; second, side B is incubated with R-LH beta, followed by species-specific secondary antibodies adsorbed in 10 nm gold particles; G anti-R IgG 10. With this technique, we demonstrated, on the same thin section, ACTH, LTH, and LH cells. The immunocytochemical procedure used has proved useful for simultaneous ultrastructural localization, on the same thin section, of three different antigenic sites. This technique, applied to other materials, could provide interesting information in several biological fields.