Development of a cryosection EM autoradiography technique and its application for the subcellular localization of receptors

1996 ◽  
Vol 54 ◽  
pp. 920-921
Author(s):  
C.-L. Na ◽  
H. K. Hagler ◽  
K. H. Muntz
Keyword(s):  
Recent Progress ◽  
Biological Molecules ◽  
Frozen Sections ◽  
Receptor Ligand ◽  
Receptor Localization ◽  
Plastic Embedding ◽  
Fresh Frozen ◽  
Em Autoradiography ◽  
Ultrathin Frozen Sections ◽  
Subcellular Level

Recent progress in immunocytochemistry and cryo-techniques has made it possible to study receptor localization at the subcellular level. For many receptor-ligand systems suitable antibodies are not available and it would be more appropriate to use radioligands to study these receptors. Although fresh frozen sections have been widely used in light microscopy (LM) autoradiography studies, to our knowledge, no one has established a technique using electron microscope (EM) autoradiography with ultrathin frozen sections.Unlike conventional EM approaches which can extract many biological molecules during dehydration and plastic embedding steps, we have adopted the method of Tokuyasu combined with LM autoradiography protocol for frozen sections to develop a new EM autoradiography technique using ultrathin frozen sections. Heart blocks were fixed in 2% periodate-lysine-paraformaldehyde (PLP) and 0.1% glutaraldehyde (GA), sucrose infused, and frozen in liquid nitrogen. They were sectioned in a Reichert Ultracut S cryo-microtome equipped with a Reichert FCS cryo-unit.

scholarly journals IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS

1971 ◽  
Vol 49 (3) ◽  
pp. 731-746 ◽  
Author(s):  
W. Bernhard ◽  
Annie Viron
Keyword(s):  
Electron Microscopy ◽  
Biological Tissues ◽  
Frozen Sections ◽  
Large Molecules ◽  
Recent Introduction ◽  
Morphological Studies ◽  
Plastic Embedding ◽  
Ultrathin Frozen Sections

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

1978 ◽  
Vol 36 (2) ◽  
pp. 90-91
Author(s):  
Kenjiro Yasuda
Keyword(s):  
Fluorescent Antibody ◽  
Pancreatic Enzymes ◽  
Tissue Preparation ◽  
Zymogen Granules ◽  
Frozen Sections ◽  
En Bloc ◽  
Antibody Method ◽  
Ultrathin Frozen Sections ◽  
Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

AUTORADIOGRAPHIC TECHNIQUES FOR THE LOCALIZATION OF HORMONES AND DRUGS AT THE CELLULAR AND SUBCELLULAR LEVEL

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S205-S222 ◽  
Author(s):  
Walter E. Stumpf
Keyword(s):  
Drug Distribution ◽  
Photographic Emulsion ◽  
Frozen Sections ◽  
Hormone Research ◽  
Freeze Dried ◽  
Subcellular Level

ABSTRACT The paper describes four autoradiographic techniques which can be recommended, not without restrictions, for the study of the cellular and subcellular hormone or drug distribution in tissues. In all of the techniques desiccated slides are used which are precoated with photographic emulsion. The techniques are (I) Dry-mounting of freeze-dried sections on emulsion precoated slides; (II) Thaw-mounting of frozen sections on emulsion precoated slides; (III) Smear-mounting on emulsion precoated slides; and (IV) Touch-mounting on emulsion precoated slides. The techniques are designed to avoid or minimize translocation of the labelled molecules during preparation and during the application to photographic emulsion. Cited examples of application of these techniques demonstrate their utility in hormone research.

scholarly journals p-HYDROQUINONE AND p-BENZOQUINONE AS HISTOCHEMICAL REAGENTS I. A NEW TETRAZOLIUM METHOD FOR AMINO GROUPS, AND THE MECHANISM OF FORMAZAN STAINING OF PARAFFIN AND FRESH FROZEN SECTIONS

1967 ◽  
Vol 15 (7) ◽  
pp. 404-408 ◽  
Author(s):  
G. G. CARMICHAEL ◽  
STEPHANIE T. K. MANDER
Keyword(s):  
Electron Acceptor ◽  
Thermal Shrinkage ◽  
Frozen Sections ◽  
Amino Groups ◽  
Paraffin Sections ◽  
Tetrazolium Bromide ◽  
Acid Ph ◽  
Reduction System ◽  
Oxidation Reduction ◽  
Fresh Frozen

The staining of amino groups by formazan when dehydrated paraffin sections are incubated in a mixture of hydroquinone and 3-(4,5-dimethyl thiazolyl-2)-2 ,5-diphenyl-2H-tetrazolium bromide at an acid pH is reported. The mechanism of this reaction and of the cytoplasmic deposition of formazan in fresh frozen sections incubated under similar conditions is investigated. It is shown that the oxidation of hydroquinone to semiquinone is responsible for the reaction, the tetrazole acting as electron acceptor. The tissue amino groups, exposed by dehydration and thermal shrinkage, and the nitrogen groupings of phosphobipid behave as "catalysts." The relevant properties of the hydroquinone-benzoquinone oxidation-reduction system are described, and the reactions between benzoquinone and tissue constituents are reviewed.

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta

1989 ◽  
Vol 257 (3) ◽  
pp. 603-607 ◽  
Author(s):  
Lopa Leach ◽  
Bryan M. Eaton ◽  
J. Anthony Firth ◽  
Soli F. Contractor
Keyword(s):  
Human Placenta ◽  
Immunoglobulin G ◽  
Frozen Sections ◽  
Immunogold Localisation ◽  
Ultrathin Frozen Sections

Effect of bacterial endotoxemia on succinic dehydrogenase of liver and kidney of rabbit

1961 ◽  
Vol 201 (1) ◽  
pp. 16-18 ◽  
Author(s):  
J. Cascarano ◽  
A. D. Rubin ◽  
A. K. Neumann ◽  
B. W. Zweifach
Keyword(s):  
Succinic Dehydrogenase ◽  
Significant Inhibition ◽  
Frozen Sections ◽  
Experimental Animals ◽  
E Coli ◽  
Fresh Frozen ◽  
Tissue Homogenates ◽  
Liver And Kidney ◽  
Lethal Doses

The in vivo inhibition of liver and kidney succinic dehydrogenase by administration of lethal doses of bacterial endotoxin ( Escherichia coli and Salmonella typhosa) was investigated. Quantitative determinations conducted on tissue homogenates revealed significant inhibition of activity only in liver of rabbits injected with E. coli lipopolysaccharide. The histochemical distribution of succinic dehydrogenase in fresh frozen sections of kidney was the same in both control and experimental animals. However, the centrolobular areas of liver appeared considerably depressed in activity in both E. coli and S. typhosa endotoxin-treated animals. These data, along with those presented by other studies in the literature, suggest that the action of endotoxin appears to be restricted to certain cells.

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