Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections

1996 ◽  
Vol 106 (3) ◽  
pp. 351-355 ◽  
Author(s):  
Ji-Ying Song ◽  
Wikky Tigchelaar ◽  
Jacques P. M. Schellens ◽  
J. Marle ◽  
C. J. F. Noorden ◽  
...  
Keyword(s):  
Low Temperature ◽  
Ultrastructural Localization ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections

1996 ◽  
Vol 106 (3) ◽  
pp. 351-355 ◽  
Author(s):  
Ji-Ying Song ◽  
Wikky Tigchelaar ◽  
Jacques P. M. Schellens ◽  
J. Van Marle ◽  
C. J. F. Van Noorden ◽  
...  
Keyword(s):  
Low Temperature ◽  
Ultrastructural Localization ◽  
Cryostat Sections ◽  
Unfixed Cryostat

scholarly journals A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

1975 ◽  
Vol 23 (3) ◽  
pp. 200-207 ◽  
Author(s):  
D M Boorsma ◽  
G L Kalsbeek
Keyword(s):  
Horseradish Peroxidase ◽  
Lupus Erythematosus ◽  
Cross Linking ◽  
Discoid Lupus Erythematosus ◽  
Gel Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Step Method ◽  
One Step ◽  
Cryostat Sections ◽  
Unfixed Cryostat

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

1966 ◽  
Vol 30 (2) ◽  
pp. 299-315 ◽  
Author(s):  
Mary G. Wetzel ◽  
Bruce K. Wetzel ◽  
Samuel S. Spicer
Keyword(s):  
Ferric Chloride ◽  
Ultrastructural Localization ◽  
Cell Types ◽  
Capillary Endothelium ◽  
Colloidal Iron ◽  
Specific Staining ◽  
Mouse Colon ◽  
Specific Localization ◽  
Cryostat Sections ◽  
Acid Mucosubstances

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.

scholarly journals Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor

2003 ◽  
Vol 51 (3) ◽  
pp. 401-404 ◽  
Author(s):  
Harald Jockusch ◽  
Sylvana Voigt ◽  
Daniel Eberhard
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Host Cells ◽  
Frozen Sections ◽  
Mouse Tissues ◽  
Transplantation Experiment ◽  
Green Fluorescent ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.

Ultrastructural localization of nicein in normal human skin by low-temperature immunoelectron microscopy

1994 ◽  
Vol 8 (1) ◽  
pp. 59
Author(s):  
T. Masunaga ◽  
H. Shimizu ◽  
A. Ishiko ◽  
D. Aberdam ◽  
J.-P. Ortonne ◽  
...  
Keyword(s):  
Low Temperature ◽  
Human Skin ◽  
Immunoelectron Microscopy ◽  
Ultrastructural Localization ◽  
Normal Human Skin ◽  
Normal Human

scholarly journals Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film.

1986 ◽  
Vol 34 (8) ◽  
pp. 1085-1094 ◽  
Author(s):  
Y Futaesaku ◽  
V Mizuhira
Keyword(s):  
Retention Rate ◽  
Cryostat Section ◽  
Ultrastructural Localization ◽  
Negative Staining ◽  
Alkaline Solutions ◽  
Gelatin Layer ◽  
Ultrastructural Damage ◽  
A New Technique ◽  
Cryostat Sections ◽  
Silver Grains

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.

A Differential Stain For Neuronal Nucleoli In Unfixed Cryostat Sections

1978 ◽  
Vol 53 (4) ◽  
pp. 195-197
Author(s):  
Pierre A. Sandoz ◽  
Elisabeth Meier
Keyword(s):  
Cryostat Sections ◽  
Unfixed Cryostat

Ultrastructural localization of carbonic anhydrase in mouse gastric parietal cells

1978 ◽  
Vol 36 (2) ◽  
pp. 532-533
Author(s):  
N. Sugai ◽  
S. Ito
Keyword(s):  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Parietal Cells ◽  
Thin Sections ◽  
Tissue Sections ◽  
Gastric Parietal Cells ◽  
Cryostat Sections ◽  
Precise Distribution

The histochemical localization of carbonic anhydrase in parietal cells has been described by a number of investigators and its presence in these cells at the ultrastructural level has been also reported. However the precise distribution of this enzyme is not clear and there are some questions regarding the validity of the histochemical reaction. In the present study, various modifications of the technique were explored and it was found that tissues fixed in buffered formaldehyde, glutaraldehyde with trinitrocresol or picric acid retained good reaction product localization of this enzyme. Cryostat sections of the fixed tissues were treated with solutions recommended by Hannson. Incubation times that were most favorable 5 to 10 min for light microscopy and 8 to 15 min for electron microscopy. For ultrastructural observations of thin sections, it was found to be important that the reacted tissue sections were post osmicated with 1% osmium tetroxide in 1. 5% potassium ferrocyanide or with aqueous 1% 0s04 for only 3 to 5 min.

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