scholarly journals Improved procedures for immunoferritin labeling of ultrathin frozen sections.

1976 ◽  
Vol 71 (3) ◽  
pp. 894-906 ◽  
Author(s):  
K T Tokuyasu ◽  
S J Singer
Keyword(s):  
Cross Linking ◽  
Frozen Sections ◽  
Glutaraldehyde Fixation ◽  
Protein Antigens ◽  
Specific Staining ◽  
Ultrathin Sections ◽  
Ultrathin Frozen Sections ◽  
Structural Preservation

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

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.

Cryo-Ultramicrotomy of Islets of Langerhans

1974 ◽  
Vol 15 (3) ◽  
pp. 591-603
Author(s):  
S. L. HOWELL ◽  
MARGARET TYHURST
Keyword(s):  
Polyethylene Glycol ◽  
B Cells ◽  
Islets Of Langerhans ◽  
Structural Features ◽  
Silicotungstic Acid ◽  
Frozen Sections ◽  
Glutaraldehyde Fixation ◽  
Ultrathin Frozen Sections ◽  
Direct Immersion ◽  
A And B Cells

A procedure is described for the preparation of ultrathin frozen sections of glutaraldehyde-fixed or unfixed islets of Langerhans by cryo-ultramicrotomy. Freezing of the tissue was accomplished by direct immersion of isolated islets in liquid nitrogen. Sectioning was performed at a specimen temperature of -80 °C and a knife temperature of -40 °C, the ribbon of sections being collected on a trough containing 60 % dimethyl sulphoxide. Staining was accomplished with 4 % silicotungstic acid and sections were protected from drying artifacts by rinsing with 0.5% polyethylene glycol. Even in tissue not subjected to prior glutaraldehyde fixation, most of the structural features of A and B cells were well preserved in frozen sections, which were obtained in a number and quality which should render them suitable for ultrastructural, cytochemical or radioautographic studies.

scholarly journals ULTRATHIN FROZEN SECTIONS

1967 ◽  
Vol 34 (3) ◽  
pp. 773-786 ◽  
Author(s):  
Elizabeth H. Leduc ◽  
W. Bernhard ◽  
S. J. Holt ◽  
J. P. Tranzer
Keyword(s):  
Enzyme Activity ◽  
Brush Border ◽  
Distal Tubule ◽  
Kidney Cortex ◽  
Mitochondrial Atpase ◽  
Frozen Sections ◽  
Enzyme Cytochemistry ◽  
Thin Sections ◽  
Ultrathin Frozen Sections ◽  
Structural Preservation

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35°C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70°C and the knife at -23°C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.

Adsorption Staining Methd For Ultrathin Frozen Sections

1980 ◽  
Vol 38 ◽  
pp. 760-763
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Negative Staining ◽  
Positive Staining ◽  
Frozen Sections ◽  
Transmission Electron ◽  
Ultrathin Sections ◽  
Immunocytochemical Studies ◽  
Ultrathin Frozen Sections

The successful routine use of cryoultramicrotomy to examine ultrathin sections by transmission electron microscopy requires the application of suitable staining to delineate the ultrastructure. While negative staining is quite effective for certain purposes1, 2, positive staining is more appropriate for immunocytochemical studies because it does not obscure the immunolabels.

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.

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:

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

scholarly journals A photocross-linking fluorescent indicator of mitochondrial membrane potential.

1993 ◽  
Vol 41 (4) ◽  
pp. 631-634 ◽  
Author(s):  
K M Hahn ◽  
P A Conrad ◽  
J C Chao ◽  
D L Taylor ◽  
A S Waggoner
Keyword(s):  
Membrane Potential ◽  
Electrical Potential ◽  
Living Cells ◽  
Covalent Attachment ◽  
Cross Linking ◽  
Mitochondrial Potential ◽  
Specific Staining ◽  
Fluorescent Indicators ◽  
3T3 Fibroblasts ◽  
Mitochondrial Staining

Ionic dyes that distribute across membranes according to electrical potential have proven valuable as fluorescent indicators of mitochondrial energetics in living cells. Applications have been limited, however, as potential-dependent staining is lost during cell fixation. We have produced a membrane potential indicator whose potential-dependent distribution can be made permanent, to enable correlation of membrane potential with cytochemical information from immunofluorescence. A carbocyanine dye was derivatized with a photoreactive nitrophenylazide moiety so that irradiation would induce nonspecific, covalent attachment to nearby molecules. Photo-induced cross-linking was observed in paper chromatography, when irradiation caused immobilization of the dye. The new dye, named PhoCy (photofixable cyanine), showed specific staining of mitochondria in living Swiss 3T3 fibroblasts. When living cells were stained, irradiated, and fixed with formaldehyde, mitochondrial staining was retained owing to cross-linking with mitochondrial components. Omission of irradiation eliminated mitochondrial staining in fixed cells. Labeling, irradiation, and fixation procedures were optimized to produce bright specific staining with minimal background. The indicator's sensitivity to mitochondrial potential was demonstrated by treating cells with 2,4-dinitrophenol, an uncoupler of mitochondrial electron transport, which decreased mitochondrial staining in living cells and in the corresponding fixed specimens.

Ultrathin frozen sections of yeast cells

1972 ◽  
Vol 40 (1-2) ◽  
pp. 197-204 ◽  
Author(s):  
Heinz Bauer ◽  
Elsje Sigarlakie
Keyword(s):  
Yeast Cells ◽  
Frozen Sections ◽  
Ultrathin Frozen Sections
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