ULTRATHIN FROZEN SECTIONS

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.

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Displacement of Acid Phosphatase and Alkaline Phosphatase Proteins in the Rat Kidney during a Dehydration Procedure. An Advantage of Ultrathin Frozen Sections for Detecting Precise Localization of an Enzyme Activity.

ACTA HISTOCHEMICA ET CYTOCHEMICA ◽

10.1267/ahc.28.143 ◽

1995 ◽

Vol 28 (2) ◽

pp. 143-148

Author(s):

Osamu Fukushima ◽

Hideki Arakawa ◽

Masaaki Kitada ◽

Masakazu Miyamura ◽

Takafumi Yatsu ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Enzyme Activity ◽

Acid Phosphatase ◽

Rat Kidney ◽

Precise Localization ◽

Frozen Sections ◽

Ultrathin Frozen Sections

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Improved procedures for immunoferritin labeling of ultrathin frozen sections.

The Journal of Cell Biology ◽

10.1083/jcb.71.3.894 ◽

1976 ◽

Vol 71 (3) ◽

pp. 894-906 ◽

Cited By ~ 177

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.

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Effects of hyper- and hypothyroidism on carbonic anhydrase, Mg2+-dependent ATPase and Mg2+-dependent, HCO3−-stimulated ATPase activities of rat duodenal mucosa and kidney cortex

Journal of Endocrinology ◽

10.1677/joe.0.1260119 ◽

1990 ◽

Vol 126 (1) ◽

pp. 119-129 ◽

Cited By ~ 6

Author(s):

S. Suzuki ◽

H. Chen ◽

T. Takahashi ◽

O. Niwa

Keyword(s):

Enzyme Activity ◽

Carbonic Anhydrase ◽

Atpase Activity ◽

Brush Border ◽

Duodenal Mucosa ◽

Kidney Cortex ◽

Serum Concentrations ◽

Dependent Atpase ◽

Brush Borders ◽

Thyroidal Status

ABSTRACT Carbonic anhydrase (CA) and Mg2+-dependent ATPase and Mg2+-dependent, HCO3−-dependent ATPase (Mg2+-HCO3−-ATPase) activities in rat duodenal mucosa and kidney cortex were examined with respect to thyroidal status. Administration of 50 and 150 μg thyroxine (T4)/kg per day s.c. for 7 days decreased duodenal cytosol CA activity to 66% of control with the former and 43% with the latter dose, while Mg2+-HCO3−-ATPase activity in brush borders of duodenal mucosa was increased to 116% of control by 150 μg T4/kg. CA and Mg2+-HCO3−-ATPase activities in the cytosol and brush border of kidney cortex did not change after administration of T4. Hypothyroidism induced by thyroidectomy for 2 and 4 weeks or administration of methimazole (2·5–20 mg/kg per day s.c. or peroral) for 2, 3 and 4 weeks all increased duodenal cytosol CA activity, to about 140% at 2 weeks and 153% at 4 weeks after thyroidectomy, and to about 136% after the oral administration of 10 mg methimazole/kg per day for 4 weeks, while brush border Mg2+-HCO3−-ATPase activity was decreased to 56% of control 4 weeks after thyroidectomy and to 74% after the s.c. administration of 20 mg methimazole/kg per day for 3 weeks. The increase in CA activity and the decrease in ATPase activity after thyroidectomy were restored to normal levels by replacement with T4. Neither enzyme activity in the kidney changed in hypothyroidism. Serum concentrations of T4 and cortisol-like material increased after administration of T4, and serum concentrations of T4, aldosterone and cortisol-like material all decreased in hypothyroidism. Correlations were observed between duodenal CA and Mg2+-HCO3−-ATPase activities and serum concentrations of T4 (P < 0·01). These results reveal that the decrease in CA activity and the increase in Mg2+-HCO3−-ATPase activity of duodenal mucosa in hyperthyroidism are reversed in hypothyroidism, while both enzyme activities in the kidney are unrelated to thyroidal status. Journal of Endocrinology (1990) 126, 119–129

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Immunoferritin localization of intracellular antigens in positively stained frozen sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008170x ◽

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).

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081310 ◽

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.

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The Application of Ultracryotomy to Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073969 ◽

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.

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Ultrathin frozen sections of yeast without aldehyde prefixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081152 ◽

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:

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Structural preservation and absolute contrast of catalase crystal sections prepared with tannic acid

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007597x ◽

1983 ◽

Vol 41 ◽

pp. 448-449

Author(s):

C.W. Akey ◽

M. Szalay ◽

S.J. Edelstein

Keyword(s):

Tannic Acid ◽

Beef Heart ◽

X Rays ◽

Screw Axis ◽

General Applicability ◽

Thin Sections ◽

Beef Liver ◽

3 Dimensional ◽

Dimensional Reconstruction ◽

Structural Preservation

Three methods of obtaining 20 Å resolution in sectioned protein crystals have recently been described. They include tannic acid fixation, low temperature embedding and grid sectioning. To be useful for 3-dimensional reconstruction thin sections must possess suitable resolution, structural fidelity and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of crystals of Pseudomonas cytochrome oxidase, orthorhombic beef liver catalase and beef heart F1-ATPase. In order to develop methods with general applicability, we have concentrated our efforts on a trigonal modification of catalase which routinely demonstrated a resolution of 40 Å. The catalase system is particularly useful since a comparison with the structure recently solved with x-rays will permit evaluation of the accuracy of 3-D reconstructions of sectioned crystals.Initially, we re-evaluated the packing of trigonal catalase crystals studied by Longley. Images of the (001) plane are of particular interest since they give a projection down the 31-screw axis in space group P3121. Images obtained by the method of Longley or by tannic acid fixation are negatively contrasted since control experiments with orthorhombic catalase plates yield negatively stained specimens with conditions used for the larger trigonal crystals.

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A combined microphysiological-computational omics approach in dietary protein evaluation

npj Science of Food ◽

10.1038/s41538-020-00082-z ◽

2020 ◽

Vol 4 (1) ◽

Author(s):

Paulus G. M. Jochems ◽

Willem R. Keusters ◽

Antoine H. P. America ◽

Pascale C. S. Rietveld ◽

Shanna Bastiaan-Net ◽

...

Keyword(s):

Enzyme Activity ◽

Brush Border ◽

Biological Effects ◽

Whey Protein Concentrate ◽

World Population ◽

Biological Efficacy ◽

Protein Sources ◽

Immune Parameters ◽

Peptide Composition ◽

Brush Border Enzyme

AbstractFood security is under increased pressure due to the ever-growing world population. To tackle this, alternative protein sources need to be evaluated for nutritional value, which requires information on digesta peptide composition in comparison to established protein sources and coupling to biological parameters. Here, a combined experimental and computational approach is presented, which compared seventeen protein sources with cow’s whey protein concentrate (WPC) as the benchmark. In vitro digestion of proteins was followed by proteomics analysis and statistical model-based clustering. Information on digesta peptide composition resulted in 3 cluster groups, primarily driven by the peptide overlap with the benchmark protein WPC. Functional protein data was then incorporated in the computational model after evaluating the effects of eighteen protein digests on intestinal barrier integrity, viability, brush border enzyme activity, and immune parameters using a bioengineered intestine as microphysiological gut system. This resulted in 6 cluster groups. Biological clustering was driven by viability, brush border enzyme activity, and significant differences in immune parameters. Finally, a combination of proteomic and biological efficacy data resulted in 5 clusters groups, driven by a combination of digesta peptide composition and biological effects. The key finding of our holistic approach is that protein source (animal, plant or alternative derived) is not a driving force behind the delivery of bioactive peptides and their biological efficacy.

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