Immunocytochemical studies of pituitary hormones with PAP, ABC, and immunogold techniques: evolution of technology to best fit the antigen.
The immunocytochemical technology in our laboratory has evolved in response to specific needs for more efficient, refined stains for each antigen. The rationale for the application of each of the immunocytochemical techniques used today is described, and detailed methods are given. In the early 1970s, it was determined that the peroxidase-antiperoxidase complex (PAP) stain provided the most sensitive means of detection of adrenocorticotropin (ACTH) at the electron microscope level even in tissues prepared with conventional fixation and embedding techniques that are considered rather harsh for the maintenance of antigenicity. Application of the same PAP complex technique to the larger glycoprotein antigens, like follicle-stimulating hormone (FSH), however, proved far more difficult; and the problem was resolved partially when more gentle fixation-embedding protocols were applied. The production of an efficient, reliable stain for FSH was achieved only with the avidin-biotin peroxidase complex (ABC) stains were applied in the early 1980s. This technique also allowed more efficient reactions for all the antigens, and morphometric data could thereby be collected more rapidly. Thus, we concluded that the light microscope immunoperoxidase techniques were excellent for the morphometric analysis of pituitary cell types in both pre-embedding and postembedding stains. However, the need for a more refined stain for its quantification at the electron microscope level on individual organelles led to the development of the colloidal gold stain in 1983-1984. This technique, which is new to our laboratory, is also described and illustrated in this report. Also included is a description of our studies of the effect of fixation and embedding processes on hormone antigenicity and techniques used to control background and nonspecific reactions. It is hoped that the novice will find the description of the rationale for the evolution of technology in our laboratory useful in making choices for his or her own immunocytochemical stains.
D000818:Animals; D001360:Avidin; D001710:Biotin; D005404:Fixatives; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006046:Gold; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D007124:Immunoenzyme Techniques; D007986:Luteinizing Hormone / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D010047:Ovalbumin / Q000031:analogs & derivatives*; D010902:Pituitary Gland / Q000378:metabolism; D010907:Pituitary Hormones / Q000276:immunology / Q000378:metabolism*; D013205:Staphylococcal Protein A
D005404:Fixatives; D010907:Pituitary Hormones; D013205:Staphylococcal Protein A; D001360:Avidin; D001710:Biotin; D006046:Gold; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D010047:Ovalbumin