Detection of endogenous and immuno-bound peroxidase — The status Quo in histochemistry

Krieg, Reimar; Kj, Halbhuber

doi:10.1016/j.proghi.2009.11.001

Cited by 27 publications

(12 citation statements)

References 169 publications

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“…Peroxidase activity (POX) in both inoculated and not inoculated leaves was evaluated qualitatively by transmission light microscopy, and quantitatively by spectrophotometry, exploiting the chromogenic property of TMB that undergoes a change in colour upon oxidation by peroxidase in presence of hydroperoxide [29,30]. …”

Section: Resultsmentioning

confidence: 99%

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

et al. 2012

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BackgroundGrapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation.ResultsThe host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated.ConclusionsSome hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

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Section: Resultsmentioning

confidence: 99%

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

et al. 2012

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“…For instance, we encountered strong background binding with different working concentrations (2-20 ug/ml) of Cy3-tyramides, made from corresponding NHS-esters that were acquired from two independent vendors (Lumiprobe and Syntol, data not shown). This background binding issue with bulky tyramides was previously mentioned by several authors, 20,21 and paradoxically, is only seen with the dye-tyramine conjugates, but not the free dyes. 21 Furthermore, we have assessed the sensitivity of CuAAC-mediated immunostaining using two different primary antibodies against NMDA receptor subunits, Glun1 and Glun2A.…”

Section: The Use Of Cuaac Reaction For Enhancement Of Immunofluorescementioning

confidence: 80%

A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining

Antonov

Новосадова

Kobylansky

et al. 2019

J Histochem Cytochem.

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The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

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“…Tyramide signal amplification (TSA) is a proprietary technique used to increase the sensitivity of detecting low numbers of antibody-antigen complexes in tissues or cells (Zaidi et al 2000;Krieg and Halbhuber 2010). The basis for activity is a time-dependent, HRP-catalyzed covalent attachment of labeled (biotin/fluorescent dye conjugated) molecules of tyramide to tyrosine residues on the nearby HRP-conjugated antibody or protein.…”

Section: Discussionmentioning

confidence: 99%

Detecting Horseradish Peroxidase-Labeled Cells

Rodig¹

2019

Cold Spring Harb Protoc

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A range of substrates is available for detection of cells labeled with horseradish peroxidase (HRP). Diaminobenzidine (DAB) is the most commonly used substrate and one of the most sensitive. It yields an intense brown product that is insoluble in both water and alcohol. DAB staining is compatible with a wide range of common histological stains. DAB can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction product is slate gray to black, and the products are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains. Alternatively, chloronaphthol gives a blue-black product and aminoethylcarbazole (AEC) yields a red product. These substrates are less sensitive than DAB and the products are soluble in alcohol, but they can be used if the DAB reaction gives too high a background or if alternative product colors are required. Diaminobenzidine (DAB) tetrahydrochloride (see Steps 1-6 and 7-13) Nickel chloride or cobalt chloride (0.3%, w/v) in H 2 O (see Steps 7-13) Tris buffer (0.05 M, pH 7.6) (for detection using DAB or chloronaphthol) Equipment Whatman No. 1 filter paper From the Antibodies collection, edited by Edward A. Greenfield.

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Detection of endogenous and immuno-bound peroxidase — The status Quo in histochemistry

Cited by 27 publications

References 169 publications

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining

Detecting Horseradish Peroxidase-Labeled Cells

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