Pre-embedding immunoelectron microscopy by peroxidase-labeled antibody method - Applications to endocrinology.

Osamura, R. Yoshiyuki; Komatsú, Noriyuki; Izumi, Shinichi; Yoshimura, Satoshi; Murakoshi, Masanori

doi:10.1267/ahc.14.383

Cited by 11 publications

(5 citation statements)

References 14 publications

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“…It is noteworthy that the immunohistochemical observations found neither the size nor the number of LH and FSH cells to change throughout the estrous cycle, that is, when the secretory granules would be released (Dada et al, 1983). Moreover electron microscopy has indicated that immunoreactivity of hormones in not only secretory granules but also in the Golgi bodies and endoplasmic reticula (Osamura et al, 1981;Nogami and Yoshimura, 1982), indicating that immunohistochemistry can detect degranulated cells. It is well known that in addition to the hormone secretory granulated cells, the parenchyma of the anterior pituitary gland contains another cell type devoid of such granules.…”

Section: Discussionmentioning

confidence: 96%

Cellular proliferation in the anterior pituitary gland of normal adult rats: Influences of sex, estrous cycle, and circadian change

Oishi¹,

et al. 1993

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Proliferative activity of the anterior pituitary gland in 10 week-old male and female rats under normal conditions was investigated by counting mitotic figures and using single and double immunostaining of 5-bromo-2'-deoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and six pituitary hormones. To determine which proliferative changes depend on the estrous cycle and circadian changes, respectively, six groups of female and two groups of male rats were studied at various times of day. Additionally, BrdU-incorporated cells were further classified by the six types of hormones they contained, or as immunonegative cells. Cell proliferative activity in the females fluctuated drastically with the highest activity in estrus and the lowest in diestrus. In the males, proliferative activity was at a relatively low level, and was similar to that in females in proestrus or early estrus, with the greater activity at night. Identified by their pituitary hormones, the distribution of the proliferating cells was almost the same in each sex, with prolactin (PRL) cells accounting for the highest proportion, followed by growth hormone (GH) cells, and adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) cells. These percentages agreed well with previously reported levels of cell types among all pituitary cells of the rat. It is therefore suggested that the life span and cycle of rat pituitary cells does not differ among cell types. In another test, male and female rats were given BrdU continuously via an osmotic pump for 8 days to compare cell proliferative activity between sexes, exclusive of the influence of estrous cycle and circadian changes. In this way, we were able to demonstrate that the cumulative incorporation of BrdU in females was consistently twice as high as in males over a constant period of time, and to conclude that cell renewal occurs at a doubled rate in the pituitary of female rat.

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Section: Discussionmentioning

confidence: 96%

Cellular proliferation in the anterior pituitary gland of normal adult rats: Influences of sex, estrous cycle, and circadian change

Oishi¹,

et al. 1993

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“…x 7,000. (Osamura et al, 1981). The localization of prolactin in condensed secretory granules of the castrated female pituitary gland is illustrated in Figure 11.…”

Section: 'mentioning

confidence: 99%

“…When the peroxidase-labeled method is used at the preembedding stage, peroxidase-labeled IgG Fab has resulted in adequate penetration into various organelles, including the doublemembrane Iimited mitochondria (Osamura et al, 1981). When the PAP method is used at preembedding, detergents such as Triton X-100 (Pickel et al, 1975) or saponin (Engehardt et al, 1984) have been utilized to improve the intracellulr penetration of the PAP complex, which is larger in molecular size than peroxidase-labeled IgG Fab.…”

Section: Introductionmentioning

confidence: 99%

“…When immunohistochemical techniques are applied a t the electron microscopic level, two different procedures are used (1) a preembedding method applied on 4-6-pm (or thicker) frozen sections (or tissue chopper sections); and (2) a postembedding method applied on ultrathin plastic sections. Recently, ultrastructural localization of antigens has been done in conjunction with various immunocytochemical principles, i.e., the peroxidase-labeled antibody method (Nakane and Pierce, 1966;Nakane, 1971Nakane, , 1975Osamura et al, 1981;Broadwell and Brightman, 1983), peroxidase-antiperoxidase (PAP) method (Sternberger, 1979), avidin-biotin peroxidase complex (ABC) technique (Childs, 1983), and other methods using heavy metals to label antibodies, i.e., ferritin or colloidal gold (Singer, 1959;Roth et al, 1981). The preembedding method can be done basically with two principles, i.e., the peroxidase-labeled antibody method or the PAP method.…”

Section: Introductionmentioning

confidence: 99%

“…In our laboratory, the preembedding method has been chosen as an immunoelectron microscopic technique because of its very stable and fine localization of various antigens in subcellular organelles, i.e., cell membrane, cytosol, rough endoplasmic reticulum, Golgi complex, secretory granules, mitochondria, and nucleus (Osamura et al, 1981;Osamura and Watanabe, 1983). When the peroxidase-labeled method is used at the preembedding stage, peroxidase-labeled IgG Fab has resulted in adequate penetration into various organelles, including the doublemembrane Iimited mitochondria (Osamura et al, 1981). When the PAP method is used at preembedding, detergents such as Triton X-100 (Pickel et al, 1975) or saponin (Engehardt et al, 1984) have been utilized to improve the intracellulr penetration of the PAP complex, which is larger in molecular size than peroxidase-labeled IgG Fab.…”

Section: Introductionmentioning

confidence: 99%

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Localization of membrane‐bound and soluble antigens using peroxidase‐labeled antibodies and preembedding immunoelectron microscopy

Osamura

1985

J. Elec. Microsc. Tech.

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The immunoelectron microscopy preembedding method using peroxidase-labeled antibodies has enabled us to localize various bioactive substances in subcellular organelles, i.e., nucleus, cytosol, perinuclear space, rough endoplasmic reticulum, Golgi complex, secretory granules, mitochondria, etc. In this text, detailed procedures and some key points for the appropriate staining are described. The applications of this technique to localize substances in various organelles and to observe ultrastructural changes of intracellular hormones are-also discussed.

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Multilayer peroxidase‐labeled antibody method: Comparison with labeled streptavidin‐biotin method, avidin‐biotin‐peroxidase complex method, and peroxidase‐antiperoxidase method

Shin

Izumi

Nakane

1995

Clinical Laboratory Analysis

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A new, extremely sensitive, "multilayer peroxidase-labeled antibody method" is introduced. This method utilizes peroxidase-labeled antibodies that immuno-react with their antigen, but also can be an antigen to other peroxidase-labeled antibodies. An example of this method is: use rabbit IgG as the first antibody, peroxidase-labeled goat antirabbit IgG as the second antibody, peroxidase-labeled rabbit antigoat IgG as the third antibody, peroxidase-labeled goat antirabbit IgG as the fourth antibody, and peroxidase-labeled rabbit antigoat IgG as the fifth antibody in succession. We compared the sensitivities of this multilayer peroxidase-labeled antibody method with that of commercially available immuno-peroxidase staining kits, i.e., avidin-biotin-complex method and peroxidase-antiperoxidase method, both by immunostaining and enzyme-linked immunosorbent assay. The generated signal and background in immunostained sections were determined by a computer-assisted image analyzer and by enzyme-linked immunosorbent assay spectrophotometrically. It was found that a maximum signal/background ratio was obtained with those sections reacted with the first four antibodies in succession by the multilayer peroxidase-labeled antibody method, and those reacted with the five antibodies in succession by the multilayer peroxidase-labeled antibody method in the enzyme-linked immunosorbent assay. Thus, the multilayer peroxidase-labeled antibody method is more sensitive than the avidin-biotin-complex method and the peroxidase-antiperoxidase method.

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Pre-embedding immunoelectron microscopy by peroxidase-labeled antibody method - Applications to endocrinology.

Cited by 11 publications

References 14 publications

Cellular proliferation in the anterior pituitary gland of normal adult rats: Influences of sex, estrous cycle, and circadian change

Cellular proliferation in the anterior pituitary gland of normal adult rats: Influences of sex, estrous cycle, and circadian change

Localization of membrane‐bound and soluble antigens using peroxidase‐labeled antibodies and preembedding immunoelectron microscopy

Multilayer peroxidase‐labeled antibody method: Comparison with labeled streptavidin‐biotin method, avidin‐biotin‐peroxidase complex method, and peroxidase‐antiperoxidase method

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