scholarly journals The labeled antigen method of immunoenzymatic staining.

1979 ◽  
Vol 27 (4) ◽  
pp. 832-840 ◽  
Author(s):  
D Y Mason ◽  
R E Sammons
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
Tissue Section ◽  
Specific Antibody ◽  
High Efficiency ◽  
Two Stage ◽  
Tissue Sections ◽  
Autoimmune Antibodies ◽  
Background Staining ◽  
Nonspecific Staining

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.

scholarly journals Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections.

1985 ◽  
Vol 33 (7) ◽  
pp. 695-698 ◽  
Author(s):  
L P Andrews ◽  
R K Clark ◽  
I Damjanov
Keyword(s):  
Monoclonal Antibody ◽  
Renal Tissue ◽  
Frozen Sections ◽  
Tissue Sections ◽  
Mouse Tissues ◽  
Background Staining ◽  
Frozen Tissue ◽  
Nonspecific Staining ◽  
Immunohistochemical Studies ◽  
Monoclonal Antibody Binding

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.

scholarly journals QUANTITATIVE ESTIMATION OF LYO- AND DESMOENZYMES IN TISSUE SECTIONS WITH AND WITHOUT FIXATION

1956 ◽  
Vol 2 (5) ◽  
pp. 487-502 ◽  
Author(s):  
Marvin M. Nachlas ◽  
William Prinn ◽  
Arnold M. Seligman
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Enzymatic Activity ◽  
Tissue Section ◽  
Salt Concentration ◽  
Leucine Aminopeptidase ◽  
Quantitative Estimation ◽  
Experimental Conditions ◽  
Tissue Sections

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, ß-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, ß-glucuronidase, and arylsulfatase, ⅓, 2/3, 2/3, ½, ⅛, and ⅛ of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

scholarly journals METHODS FOR THE STUDY OF SMALL PHAGOSOMES AND THEIR RELATIONSHIP TO LYSOSOMES WITH HORSERADISH PEROXIDASE AS A "MARKER PROTEIN"

1967 ◽  
Vol 15 (7) ◽  
pp. 375-380 ◽  
Author(s):  
WERNER STRAUS
Keyword(s):  
Acid Phosphatase ◽  
Low Temperature ◽  
Horseradish Peroxidase ◽  
Acid Medium ◽  
Tissue Section ◽  
Double Staining ◽  
Marker Protein ◽  
Polar Media ◽  
The Stability ◽  
Blue Reaction

Small phagosomes (micropinocytic vesicles and vacuoles) which had taken up injected horseradish peroxidase were identified by staining for peroxidase with benzidine and H2O2. Because of the small size of the granules and the possibility of artifacts, previously described procedures had to be modified in several respects. Prefixation of the tissue by perfusion at 37°C prevented artifacts of diffusion and adsorption of peroxidase. The blue product of the reaction of peroxidase with benzidine in the small phagosomes was preserved and fading to brown was prevented by cooling the tissue section to –10° to –15°C during its processing through polar media. The blue reaction product was stable as soon as the section was transferred to an apolar medium. Small phagosomes were visualized together with lysosomes and phago-lysosomes in the same cells by double staining for acid phosphatase and peroxidase in contrasting colors. The incubation for acid phosphatase was performed at 4°C since low temperature increased the stability of peroxidase in the acid medium. Factors which form the basis for other improvements of the procedure are discussed.

High-efficiency CuInSe2 solar cells prepared by the two-stage process

Solar Cells ◽  
1989 ◽  
Vol 27 (1-4) ◽  
pp. 299-306 ◽  
Author(s):  
Bulent M. Basol ◽  
Vijay K. Kapur ◽  
Richard C. Kullberg
Keyword(s):  
Solar Cells ◽  
High Efficiency ◽  
Two Stage ◽  
Stage Process

scholarly journals Development of high efficiency 4 K two-stage pulse tube cryocoolers with split valve unit

2012 ◽  
Author(s):  
Kyosuke Nakano ◽  
Mingyao Xu ◽  
Hirokazu Takayama ◽  
Akihiro Tsuchiya ◽  
Motokazu Saito
Keyword(s):  
High Efficiency ◽  
Pulse Tube ◽  
Two Stage ◽  
Valve Unit

Visualisation of GABAergic neurons and synapses in the rat brain using immunohistochemistry for two forms of glutamate decarboxylase

2021 ◽  
Vol 21 (2) ◽  
pp. 63-73
Author(s):  
Valeria A. Razenkova ◽  
Dmitrii E. Korzhevskii
Keyword(s):  
Brain Tissue ◽  
Glutamate Decarboxylase ◽  
Brain Structures ◽  
Gabaergic Neurons ◽  
Laboratory Animals ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Background Staining ◽  
The Central Nervous System ◽  
Rat Forebrain

BACKGROUND: Taking into account the importance of GABAergic brain system research and also the opportunity to achieve specific and accurate results in laboratory studies using immunohistochemical approaches, it seems important to have a reliable method of visualization GABA-synthesizing cells, their projections and synapses, for the morphofunctional analysis of GABAergic system both in normal conditions and in the experimental pathology. AIM: The aim of the study was to visualize analyze GABAergic neurons and synapses within rats brain using three different antibody types against glutamate decarboxylase and to identify the optimal conditions for reaction performing. MATERIALS AND METHODS: The study was performed on paraffin brain tissue sections of 5 adult Wistar rats. Immunohistochemical reactions using three antibody types against glutamate decarboxylase isoform 67 (GAD67) and glutamate decarboxylase isoform 65 (GAD65) were performed. Additional controls on C57/Bl6 mice and Chinchilla rabbits brain samples were also carried out. RESULTS: Antibodies used in the research made it possible to achieve high quality of GABAergic structures visualizing without increasing background staining. At the same time different antibody types are distinct in their efficacy to perform immunohistochemistry reaction on laboratory animal brain tissue samples. By performing additional controls, we discovered that there is necessary to adsorb secondary reagents immunoglobulins in order to eliminate nonspecific staining. It was found that GAD67 and GAD65 distribution in rat forebrain structures is different. It was stated that GAD67 immunohistochemistry most completely reveals GABAergic brain structures compared to GAD65 immunhistochemistry. The possibility of determining morphological features of GABAergic neurons and synaptic terminals, as well as performing quantitative analysis, was demonstrated. CONCLUSIONS: The approach proposed makes it possible to specifically visualize GABAergic structures of the central nervous system of different laboratory animals. This could be useful both in fundamental studies and in pathology research.

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