Application of the avidin-biotin-peroxidase complex (ABC) method to the light microscopic localization of pituitary...

The avidin-biotin-peroxidase complex (ABC) method was applied to semithin (0.5-1 micron) plastic-embedded sections of intact male rat pituitaries with the use of techniques previously developed for the peroxidase-antiperoxidase complex (PAP) method. Stains for adrenocorticotropin (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were cleaner, more reliable, and more efficient. The ABC method allowed the use of the same high dilutions of primary antisera used with the PAP method. Incubation time was cut to a third of the time used for the PAP stain. Furthermore, if the incubation time matched that used with the PAP method, (24-48 hr), the antisera could be diluted 2- to 4-fold further. This enhanced specific staining and allowed the use of dilutions similar to those used in the radioimmunoassay. In agreement with Hsu and Raine (J Histochem Cytochem 29:1349, 1981), the ABC method produced staining after only a 1-4 hr incubation in primary antibody that was diluted optimally for the PAP complex method. The stain was weak, however, and cell counts showed that it was restricted to the fraction of the specific cell population which stored the most hormone. Our tests showed that the most convenient incubation times for optimal staining were 12-16 hr. Furthermore, the ABC method appeared to stabilize greatly the reaction for FSH and thus improved its precision and reliability.