Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method.

doi:10.1177/18.3.161

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Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method.

Journal Article•DOI•

Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method.

01 Mar 1970-Journal of Histochemistry and Cytochemistry (SAGE Publications)-Vol. 18, Iss: 3, pp 161-166

TL;DR: Peroxidase-labeled antibody method was used to localize tissue antigens directly on ultrathin sections of methacrylate-embedded anterior pituitary gland of the rat and totally eliminates the problem of penetration of antisera through tissues and permits better immunologically controlled experiments by using serially sectioned materials.

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Abstract: 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.

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Journal Article•DOI•

Immunochemistry on ultrathin frozen sections

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K. T. Tokuyasu¹•Institutions (1)

University of California, San Diego¹

01 Jul 1980-Histochemical Journal

TL;DR: It is considered that cryoultramicrotomy has reached the stage of being useful in studying many questions which will not be easily approached otherwise, and is considered to be useful for immunochemical localization of intracellular antigens.

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Abstract: Ultrathin frozen sections can be cut smoothly from many fixed and appropriately treated specimens. To use such sections for immunochemical localization of intracellular antigens, fixa ion conditions must be selected to optimize at least three variables, namely, preservation of ultrastructure, preservation of antigenicity and retention of accessibility of the antigen to the antibody. Furthermore, staining of the sections must be such that both the immunolabels and structures are clearly recognized. Our efforts to attain these goals are described in relation to their historical background. Although there are still problems to be solved and improvements to be made, we now consider that cryoultramicrotomy has reached the stage of being useful in studying many questions which will not be easily approached otherwise.

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678 citations

Journal Article•DOI•

Adenohypophysis: ultrastructural cytochemistry. A review.

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Gwen C. Moriarty

01 Oct 1973-Journal of Histochemistry and Cytochemistry

300 citations

Cites background or methods from "Localization of tissue antigens on ..."

...Nakane localized prolactin in rats at the electron microscopic level with antirat prolactin and antiimmunoglobulin peroxidase conjugates (57, 92, 93)....
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...This may have been due to a lower sensitivity of the ferritin antibody stain, since Nakane has described some slight staining for GH in the rough endoplasmic reticulum and Golgi cisternae (57, 92, 93)....
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...Nakane used antiovine FSH to localize FSH cells in rats (92) and anti-HCG to localize LH (57, 92, 93, 136)....
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...Immunoenzyme techniques were first described in 1966 (3, 94), and Nakane was the first to use an immunoperoxidase technique to localize pituitary hormones at the light and electron microscopic level (57, 75, 91-93, 136)....
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...He stained GH cells which were embedded in methacrylate (57, 92) or Epon (93)....
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Journal Article•DOI•

Cellular localization of tyrosine hydroxylase by immunohistochemistry.

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Virginia M. Pickel, Tong H. Joh, P M Field, C G Becker, Donald J. Reis - Show less +1 more

01 Jan 1975-Journal of Histochemistry and Cytochemistry

TL;DR: The perikarya of noradrenergic neurons could be distinguished from dopaminergic neurons by the immunohistochemical demonstration of the enzyme dopamine-beta-hydroxylase only in the former.

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Abstract: The enzyme tyrosine hydroxylase (TH) was immunohistochemically localized by the peroxidase-antiperoxidase method in rat to chromaffin cells of the adrenal medulla, large neurons and small darkly staining cells of the superior cervical ganglia and noradrenergic and dopaminergic neurons in brain. As compared with the conjugated peroxidase or immunofluorescence techniques, the peroxidase-antiperoxidase method gave the most selective and specific cytoplasmic localization of TH antisera in every tissue examined. The peroxidase staining with the TH antisera was more intense in dopaminergic than in noradrenergic neurons of the central nervous system. While TH was visualized in cell bodies of both dopaminergic and noradrenergic neurons, it could only be detected in axons and terminals in the dopaminergic system. The perikarya of noradrenergic neurons could be distinguished from dopaminergic neurons by the immunohistochemical demonstration of the enzyme dopamine-beta-hydroxylase only in the former.

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257 citations

Cites background from "Localization of tissue antigens on ..."

...and then reacted with conjugated peroxidase for 30 mm (21)....
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Journal Article•DOI•

Recent progress in the peroxidase-labeled antibody method.

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Paul K. Nakane¹•Institutions (1)

University of Colorado Boulder¹

01 Jun 1975-Annals of the New York Academy of Sciences

TL;DR: Improvements in the methodologies associated with the conjugation of peroxidase to immunoglobulin, the fixation of antigens, and the penetration of peroxin-labeled antibodies into tissues should greatly facilitate successful application of the peroxidaselabeled antibody method.

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Abstract: Almost a decade has passed since the initial application of enzymes as labels in immunohistocytochemistry. During these years, the peroxidase-labeled antibody method has been used successfully by several laboratories for localizing antigens at both the light and electron microscopic levels. Recent improvements in the methodologies associated with the conjugation of peroxidase to immunoglobulin, the fixation of antigens, and the penetration of peroxidaselabeled antibodies into tissues should greatly facilitate successful application of the method.

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245 citations

Journal Article•DOI•

Electron microscopic study of the adrenocorticotropin-producing cell with the use of unlabeled antibody and the soluble peroxidase-antiperoxidase complex.

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G. C. Moriarty, N. S. Halmi

01 Aug 1972-Journal of Histochemistry and Cytochemistry

TL;DR: Twenty-one days after adrenalectomy, the ACTH cells are greatly increased in numbers and have complex, tortuous processes filled with intensely stained secretory granules, suggesting that an early effect of cortisol is to block release of ACTH.

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Abstract: The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of c

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220 citations

Cites background from "Localization of tissue antigens on ..."

...Nakane PK: Application of peroxidase-labelled antibodies to the intracellular localization of hormones....
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...For this purpose, Kawarai and Nakane (15, 23) developed a “steady flow” apparatus in which a steady stream of the solution was pumped over the grids attached to a glass slide....
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...Nakane PK, Pierce GB Jr: Enzyme-labeled antibodies: preparation and application for the localization of antigens....
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...Nakane has used peroxidase-labeled anti- bodies to localize ACTH at the electron microscopic level (22, 23)....
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...Nakane noted that the ACTH cells were difficult to tell ultrastructurally from thyrotrophs without the use of the immunohistochemical stain....
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References

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Journal Article•DOI•

The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique.

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Richard C. Graham, Morris J. Karnovsky

01 Apr 1966-Journal of Histochemistry and Cytochemistry

TL;DR: The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique, which gives sharp localization and is sensitive to protein transport.

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Abstract: The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique. In animals killed as early as 90 sec after injection, reaction product was found on the brushborder membranes and in the apical tubular invaginations. From the latter structures it was transported to the apical vacuoles, in which it was progressively concentrated to form protein absorption droplets. The method, which employs 3,3'-diaminobenzidine as oxidizable substrate, gives sharp localization and is sensitive. This system is advantageous in studying the early stages of renal tubular protein absorption, since small amounts of protein on membranes and in tubules and vesicles can be detected easily. The method also appears promising for studying protein transport in a variety of other cells and tissues.

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6,495 citations

"Localization of tissue antigens on ..." refers background or methods in this paper

...It was found that about one-tenth of the concentration of substrate medium developed by Graham and Karnovsky (2) gave satisfactory results....
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...Karnovsky, M. J.: Simple method for “staining with lead” at high pH in electron microscopy....
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...Graham, R. C. and Karnovsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique....
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...Initially the sections were incubated on a droplet of incubation media using 3,3’-diaminobenzidene as substrate for the enzyme (2), but enough reaction product was deposited in nonspecific manner on the surfaces of the section to obscure the specific...
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Book•

Fluorescent antibody methods

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Coons Ah

01 Jan 1968

1,594 citations

Journal Article•DOI•

Simple methods for "staining with lead" at high pH in electron microscopy.

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Morris J. Karnovsky¹•Institutions (1)

Harvard University¹

01 Dec 1961-Journal of Cell Biology

TL;DR: It is thought that in these highly alkaline staining solutions lead is present as an hydroxide complex anion (plumbite ion) and that this anion is responsible for the staining, and the methods of preparation are based on this hypothesis.

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Abstract: The lead hydroxide stain of Watson (1958) used for increasing contrast in thin sections for electron microscopy has found acceptance in many laboratories. However, this stain has an unfortunate tendency to form precipitates (probably of lead carbonate (5)) on exposure to the air, thus contaminating the sections and irritating the observer. This drawback has led to the development of several modifications (2, 3) of the original method of staining and the use of ingenious devices (4, 5) for preventing exposure to air and consequent precipitate formation. We offer the following alternative methods which, we believe, are simpler to perform than those hitherto described. They have the additional advantages mentioned below. The methods are based on the observation that highly alkaline solutions of lead salts (pH > 11.5) yield relatively stable solutions which stain rapidly and intensely, thus obviating the hazard of precipitation to a marked degree. The methods have these additional advantages: the staining solutions are easily and rapidly prepared, are simply stored, and are stable for long periods of time. Furthermore, they can be efficiently used, many grids being treated simultaneously, without excessive precautions being taken against lead carbonate precipitation. Finally, \"difficult\" material, embedded in media which characteristically yield rather low contrast, such as epoxide resins, can be rapidly and easily stained. \"C lean\" preparations, of high contrast, are routinely obtained. As will be discussed later, it is thought that in these highly alkaline staining solutions lead is present as an hydroxide complex anion (plumbite ion) and that this anion is responsible for the staining. The methods of preparation are based on this hypothesis. Two methods for preparing the staining solutions have been found useful:

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1,298 citations

"Localization of tissue antigens on ..." refers methods in this paper

...The sections were observed with or without counterstaining with lead hydroxide (3) under JEM 100 B electron microscopes....
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Journal Article•DOI•

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

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Nakane Pk, Pierce Gb

01 Dec 1966-Journal of Histochemistry and Cytochemistry

TL;DR: The peroxidase was conjugated to antibody by a method similar to one developed for the comijugations of ferritimi to auitibody, and the best amid most consistent resumlts were obtained when FNPS was umsed.

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Abstract: 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-

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1,005 citations

"Localization of tissue antigens on ..." refers methods in this paper

...Rabbit antihuman chorionic gonadotropin, which crossreacts specifically with rat luteinizing hormone (6), rabbit anti-rat growth hormone, rabbit antirat prolactin and peroxidase-labeled sheep antirabbit -y-globulin (9, 10) were used....
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...These were placed on a glass slide, the embedding medium was removed by 100% xylene and the specimens were stained immunohistochemically by the usual means (9)....
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...The peroxidase-labeled antibody method has been used to localize tissue antigens both at light and electron microscopic levels (4, 7-10)....
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