Paul K. Nakane

PERIODATE-LYSINE-PARAFORMALDEHYDE FIXATIVE A NEW FIXATIVE FOR IMMUNOELECTRON MICROSCOPY

A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue preservation, a task which has not been successfully accomplished by conventional fixatives.

PEROXIDASE-LABELED ANTIBODY A NEW METHOD OF CONJUGATION'

A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with sodium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immunoglobulin retained its immunologic as well as enzymatic activities.

Enzyme-labeled antibodies: preparation and application for the localization of antigens.

primiciples of immummiohiistochemist ry to electron niicroscopy have niet with very limited sumccess (Pierce et at., mt. Rev. Exp. Path. 3: 1. 1964). Ins ami effort to obviate somuie of these himnitatiomus, tissumes have beemt staimied with emszymes conujumgated to antibody. Ins the imuitil experintemits, acid phosphsatase was comijugated to antibody (Ram et al., Fed. Proc. 25: 732. 1966). Although these preliminary experiments deniomustrated the feasibility of thie approachu, the resumlts of the couijugatiomss were so variable that search was made for a better enzyme. Imu the presemut stumdy horseradish peroxidase was employed becaumse the enzynte is commercially available ims relatively pure form; the cytochemical amid histochemical methods have been well established, and successfully adapted to electron microscopy (Graham amid Karnovsky, J. Histochem. (Jytochem. 14: 291. 1966). The peroxidase was conjugated to antibody by a method similar to one developed for the comijugations of ferritimi to auitibody (Ram et at., J. Cell Biol. 17: 673. 1963), amid acid phosphiatase to amitibody (Ram et at., Fed. Proc. 25: 732. 1966; Nakane et at., J. Histochein. Cytochem. 7th Anmuumal Meetimig, Abstract, 1966), which employed the bifummictional reagent, p,p’difiumoro-m-m’-diuiitrodiphenyl sulfomie (FNPS). Other bifummuctiomual reagemuts sucht as 1-ethiyl-3-(3dimethylanuino propyl) carbodiimide have also beemi employed, bunt the best amid most consistent resumlts were obtained when FNPS was umsed. For routimie stumdies the conjumgates were pre-

SIMULTANEOUS LOCALIZATION OF MULTIPLE TISSUE ANTIGENS USING THE PEROXIDASE-LABELED ANTIBODY METHOD: A STUDY ON PITUITARY GLANDS OF THE RAT

The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section. The first antigen was localized by the indirect method and the antisera were removed from the section by elution, leaving the colored reaction products identifying the antigenic sites. The second and subsequent antigens were localized similarly, using substrates that develop reaction products of different colors. For the demonstration of the method, anti-human growth hormone (GH), anti-human thyrotropic hormone (TSH) and anti-human chorionic gonadotropin, which cross-reacts with rat luteinizing hormone (LH), were used with sections of pituitary gland of the rat. Anti-GH reacted with small acidophilic cells. Anti-LH was localized in oval shaped cells, and anti-TSH was localized in angulated basophilic cells. Some cells were devoid of these three antigens and no cell contained more than one of them.

CLASSIFICATIONS OF ANTERIOR PITUITARY CELL TYPES WITH IMMUNOENZYME HISTOCHEMISTRY

Peroxidase-labeled antibody method was used to localize the six hormones of the anterior pituitary gland of male rats both at the light and electron microscopic levels. Growth hormone (GH), adenocorticotropic hormone (ACTH), prolactin and thyrotropic hormone (TSH) were found in separate cells. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were frequently found in the same cell. TSH cells were scarce and were located at the periphery of the gland. The anterior-ventral portion of the gland contained few or no GH cells, ACTH cells, prolactin cells and TSH cells, but was filled with gonadotropic cells. In an area near the intermediate lobe, GH cells, ACTH cells and TSH cells were not found. GH cells and prolactin cells may be identified in electron micrographs without the aid of immunocytochemistry; however, ACTH cells and TSH cells may not be distinguished by their ultrastructural characteristics alone. Gonadotropic cells may be identified but their hormone content cannot be determined. The positive identification of these latter four cell types requires immunocytochemical methods.

Studies on Translocation of Immunoglobulins Across Intestinal Epithelium: II. Immunoelectron-microscopic localization of immunoglobulins and secretory component in human intestinal mucosa

To define mechanisms involved in the transport of immunoglobulins into intestinal fluids, we localized IgM, IgA, IgG, and secretory component (SCI in human intestinal mucosa by the peroxidase-labeled antibody technique. At the light microscopic level, immunocytes containing IgA, IgM, or IgG were found in the lamina propria. IgA, IgM, and SC were prominent in the epithelium of gland crypts; IgG was limited to a few cells at tips of villi. At the electron-microscopic level, SC was localized to perinuclear spaces, endoplasmic reticulum, saccules associated with Golgi complexes, cytoplasmic vesicles, and lateral and basal plasma membranes of columnar epithelial cells. IgA and IgM, but not IgG, also were localized to plasma membranes and cytoplasmic vesicles of these cells. Neither the immunoglobulins nor SC was found within other types of epithelial cells (Paneth, goblet, endocrine). The findings provide evidence that (1) the site of SC synthesis in intestinal epithelium is secretory columnar cells, principally those in gland crypts; (2) the polymeric immunoglobulins IgM and IgA are translocated through such SC-containing cells by a process that involves formation of cytoplasmic vesicles; (3) IgM and IgA could combine with SC during transcellular transport (likely sites are lateral or basal plasma membranes or supranuclear cytoplasm); (4) the monomeric immunoglobulin IgG does not share the transepithelial cell route involved in IgM and IgA transport.

LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF ANTIGENS IN TISSUES EMBEDDED IN POLYETHYLENE GLYCOL WITH A PEROXIDASE-LABELED ANTIBODY METHOD

Tissues embedded in polyethylene glycol (PEG) were used for the immunocytochemical localization of cellular antigens at the light and electron microscopic level. For demonstration of the method, growth hormone and luteinizing hormone were localized in the anterior pituitary gland of the rat embedded in PEG, using peroxidase-labeled antibodies. PEG is water-soluble, has a low melting temperature yet is firm enough to be sectioned at room temperature. Preservation of cellular ultrastructure is good. The use of PEG-embedded tissue permits precise correlation of light and electron microscopic observations of the same tissue section.

LOCALIZATION OF TISSUE ANTIGENS ON THE ULTRATHIN SECTIONS WITH PEROXIDASE-LABELED ANTIBODY METHOD

Peroxidase-labeled antibody method was used to localize tissue antigens directly on ultrathin sections. For the demonstration of the method, luteinizing hormone, growth hormone and prolactin were localized on ultrathin sections of methacrylate-embedded anterior pituitary gland of the rat. The hormones were localized in secretion granules and in endoplasmic reticulum, but not in nucleus or mitochondria. This approach totally eliminates the problem of penetration of antisera through tissues and permits better immunologically controlled experiments by using serially sectioned materials.

Effect of Thyrotropin-Releasing Factor on Thyrotropic Cells in vitro*

Publisher Summary This chapter presents the effect of thyrotropin-releasing factor (TRF) on the thyrotropic cells [thyrotropic hormone (TSH) cells] that was investigated in vitro , and it was found that the TSH cells undergo degranulation rapidly following the exposure to the TRF in vitro. TSH cells were more frequently found in clusters in the center of the gland. The cells were polygonal to stellate in shape and usually located in the center of the cord. Signs of TSH release were noticed in those pituitary glands that were placed in the incubation medium immediately after the removal from the animals. After five minutes of exposure of the gland to TRF, some TSH cells were degranulated, their cytoplasm filled with hydropic vacuoles, and anti-TSH positive granules found among the vacuoles. In conclusion, the synthetic TRF is capable of releasing the TSH cells in vitro. The morphological changes associated with the release of TSH are similar to those observed in other cell types, and it may be concluded that the phase of the release of the pituitary hormones from the pituitary glands are very similar.