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 CYTOCHEMICAL LOCALIZATION OF ADENYL CYCLASE ACTIVITY IN RAT ISLETS OF LANGERHANS

1972 ◽  
Vol 20 (11) ◽  
pp. 873-879 ◽  
Author(s):  
S. L. HOWELL ◽  
MARGARET WHITFIELD
Keyword(s):  
B Cells ◽  
Islets Of Langerhans ◽  
Adenosine Triphosphate ◽  
Adenyl Cyclase ◽  
Cyclase Activity ◽  
Cytochemical Localization ◽  
Adenyl Cyclase Activity ◽  
Rat Islets ◽  
Rat Islets Of Langerhans ◽  
A And B Cells

A cytochemical method has been used to investigate the localization of adenyl cyclase activity in A and B cells of isolated rat islets of Langerhans. Adenosine triphosphate was initially utilized as substrate, the pyrophosphate liberated being precipitated by lead ions at its site of production. The specificity of the method was increased by the use of adenylyl-imidodiphosphate as an alternative substrate; this adenosine triphosphate analogue was not hydrolyzed by adenosine triphosphatase but provided an effective substrate for adenyl cyclase. Adenyl cyclase activity, which was found to retain its glucagon and fluoride sensitivity in glutaraldehyde-fixed tissue, was found exclusively and almost uniformly in the plasma membranes of A and B cells. Storage granule membrane, incorporated into the plasma membrane during secretion of the granule content by exocytosis, appeared to be devoid of adenyl cyclase activity.

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.

scholarly journals Biochemical and ultrastructural changes in A and B cells of the islets of Langerhans of mice infected with EMC virus

Diabetologia ◽  
1979 ◽  
Vol 17 (1) ◽  
pp. 51-57 ◽  
Author(s):  
F. Zaheer ◽  
S. L. Howell ◽  
K. W. Taylor ◽  
T. J. Coleman ◽  
D. R. Gamble
Keyword(s):  
B Cells ◽  
Islets Of Langerhans ◽  
Ultrastructural Changes ◽  
Emc Virus ◽  
A And B Cells

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:

scholarly journals Characterization of voltage-dependent sodium and calcium channels in mouse pancreatic A- and B-cells

2006 ◽  
Vol 572 (3) ◽  
pp. 691-706 ◽  
Author(s):  
Sheila Vignali ◽  
Veronika Leiss ◽  
Rosi Karl ◽  
Franz Hofmann ◽  
Andrea Welling
Keyword(s):  
B Cells ◽  
Calcium Channels ◽  
Voltage Dependent ◽  
A And B Cells
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