scholarly journals Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach.

1989 ◽  
Vol 37 (4) ◽  
pp. 473-477 ◽  
Author(s):  
K W Schmid ◽  
A Hittmair ◽  
H Schmidhammer ◽  
B Jasani
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Sodium Azide ◽  
Inhibitory Action ◽  
Selective Inhibition ◽  
Surface Antigens ◽  
Immunoperoxidase Staining ◽  
Human Tonsil ◽  
Maximum Inhibition ◽  
Endogenous Peroxidase

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.

scholarly journals Factors affecting the sensitivity and specificity of the cytochemical reaction for the anti-horseradish peroxidase antibody in lymph tissue sections.

1980 ◽  
Vol 28 (7) ◽  
pp. 645-652 ◽  
Author(s):  
W Straus
Keyword(s):  
Horseradish Peroxidase ◽  
Lymph Nodes ◽  
Sensitivity And Specificity ◽  
Peroxidase Activity ◽  
Additional Advantage ◽  
Tissue Sections ◽  
Endogenous Peroxidase ◽  
Cytochemical Reaction ◽  
Cold Acetone ◽  
Antibody Reaction

Factors which increase the sensitivity and specificity of the cytochemical reaction for the antibody to horseradish peroxidase (HRP) in precursors of plasma cells and in lymphocytes were studied in sections of popliteal lymph nodes of rats. The lymph nodes were removed 3-5 days after a secondary injection of HRP into the footpads and were fixed for 5 hr in a 4% cold formaldehyde solution (Straus W: Histochemistry 53:273, 1977). Brief postfixation of the frozen sections with cold acetone improved the retention of the antigen at the sites of the antibody in the precursor cells, and it improved the quality of fixation without appreciably weakening the antigen-binding capacity of the antibody. The cytochemical reaction for the anti-HRP antibody was intensified by staining with diaminobenzidine (DAB) and H2O2 at pH 5-6, or by staining at pH 7.4 in the presence of imidazole. Imidazole partially inhibited endogenous peroxidase activity. Pretreatment with phenylhydrazine prevented nonspecific background adsorption of HRP. Phenylhydrazine had the additional advantage of inhibiting most of the endogenous peroxidase activity (Straus W: J Histochem Cytochem 20:949, 1972). The intensity of the antibody reaction in the proplasma cells developing in the medullary cords varied greatly depending on the stage of maturation from lymphocytes and blast cells. Many lymphocytes in the cortex of the lymph node showed a strong perinuclear antibody reaction when the tissue sections were postfixed with cold acetone, and the peroxidase complexed to the antibody was visualized by staining with DAB and H2O2 at pH 5-6. The antibody reaction also occurred at the surgace of many lymphocytes when the tissue sections, postfixed with cold acetone, were stained with DAB and H2O2 at pH 7.4 in the presence of imidazole. Other lymphocytes showed a strong surface, perinuclear, and cytoplasmic antibody reaction after staining at pH 5-6 as well as after staining at pH 7.4, while yet other lymphocytes remained unstained.

scholarly journals INHIBITION OF PEROXIDASE BY METHANOL AND BY METHANOL-NITROFERRICYANIDE FOR USE IN IMMUNOPEROXIDASE PROCEDURES

1971 ◽  
Vol 19 (11) ◽  
pp. 682-688 ◽  
Author(s):  
WERNER STRAUS
Keyword(s):  
Acetic Acid ◽  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Considerable Extent ◽  
Absolute Methanol ◽  
Endogenous Peroxidase ◽  
Cytochemical Reaction ◽  
The Absolute

The cytochemical reaction for peroxidase is partially inhibited by methanol. The addition of a small amount of sodium nitroferricyanide to the absolute methanol causes further inhibition. The activity of peroxidase is completely suppressed by methanol containing 1% sodium nitroferricyanide and l% acetic acid. The antibody to horseradish peroxidase is much less affected by these inhibitors than horseradish peroxidase itself. By using appropriate combinations of these inhibitors and by varying the time or temperature of treatment, the activity of injected horseradish peroxidase, or of endogenous peroxidase in leukocytes, erythrocytes or normoblasts, can be much decreased or suppressed while the specific ability of the antibody to adsorb horseradish peroxidase can be preserved to a considerable extent. KCN and NaN3 inhibit peroxidase activity only moderately under conditions required for immunoperoxidase procedures, i.e., after removal of the inhibitors.

scholarly journals Endogenous peroxidase activity in human cutaneous and adenoidal mast cells.

1987 ◽  
Vol 35 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L M Escribano ◽  
L C Gabriel ◽  
E Villa ◽  
J L Navarro
Keyword(s):  
Endoplasmic Reticulum ◽  
Mast Cells ◽  
Golgi Apparatus ◽  
Peroxidase Activity ◽  
Incubation Time ◽  
Sodium Azide ◽  
Incubation Medium ◽  
Potassium Cyanide ◽  
Sodium Pyruvate ◽  
Endogenous Peroxidase

We have studied peroxidase activity in human cutaneous and adenoidal mast cells using different methods, in order to determine the optimal technical conditions for its demonstration. In 1.25% glutaraldehyde-fixed cells, no peroxidase activity was seen. On the contrary, in tannic acid-aldehyde-fixed cells or in unfixed cells peroxidase activity was revealed independently of the DAB concentration or the incubation time in DAB medium. The reaction product was localized in perinuclear cisternae and endoplasmic reticulum. Granules were always unreactive with all techniques employed. Golgi apparatus was generally negative and only occasional cells exhibited one or two positive peripheral cisternae. This activity appears sensitive to fixation by glutaraldehyde and is inhibited by 3-amino-1,2,4-triazole (AMT) and by lack of H2O2 or DAB in the incubation medium, but not by potassium cyanide, sodium azide, or sodium pyruvate, at the concentrations used. The peroxidase activity described in this report is an endogenous peroxidase and is not related to uptake of exogenous peroxidase by mast cells. It can therefore be considered as an ultracytochemical marker of human mast cells.

Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin.

1976 ◽  
Vol 22 (8) ◽  
pp. 1372-1377 ◽  
Author(s):  
D E Yorde ◽  
E A Sasse ◽  
T Y Wang ◽  
R O Hussa ◽  
J C Garancis
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Quantitative Measurement ◽  
Original Sample ◽  
Soluble Enzyme ◽  
Soluble Complex ◽  
Human Choriogonadotropin ◽  
Preliminary Study ◽  
Free Antigen ◽  
Serum And Urine

Abstract We described the principle of a new enzyme-immunoassay, competitive enzyme-liked immunoassay (CELIA), for quantitative measurement of soluble antigens and haptens. In the assay, binding of antibody to antigen-immunosorbent is competitively inhibited by the free antigen to be measured. The amount of first antibody bound to the immunosorbent is measured by an enzymatic technique in which a heterologous bridging antibody and a soluble antibody/enzyme immune complex are applied in sequence. The soluble complex we used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the concentration in the original sample of the substance to be assayed. The enzyme-linked reagents are potentially widely applicable to any substance to be measured. To demonstrate the feasibility of CELIA, we report a preliminary study of its application to the measurement of human chloriogonadotropin in serum and urine. The assay described for this hormone has a working range of 1 to 50 int. units per milliliter of sample. The technique obviates the disadvantages associated with measurement and handling of radioisotopes in radioimmunoassays and the only major instrumentation required is a centrifuge and a conventional spectrophotometer.

scholarly journals Acidic horseradish peroxidase activity abolishes genotoxicity of common dyes

2017 ◽  
Vol 321 ◽  
pp. 576-585 ◽  
Author(s):  
Barbara S. Janović ◽  
Andrew R. Collins ◽  
Zoran M. Vujčić ◽  
Miroslava T. Vujčić
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity

scholarly journals DEVELOPMENT OF ENDOGENOUS PEROXIDASE IN FETAL RAT SUBMANDIBULAR GLAND

1973 ◽  
Vol 21 (1) ◽  
pp. 42-50 ◽  
Author(s):  
SHOHEI YAMASHINA ◽  
TIBOR BARKA
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Submandibular Gland ◽  
Rough Endoplasmic Reticulum ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Prenatal Development ◽  
Reaction Products ◽  
Cell Type ◽  
Endogenous Peroxidase

The prenatal development of endogenous peroxidase activity in the submandibular gland of rat was investigated by means of the diaminobenzidine-H2O2 histochemical method. The submandibular gland of a 16-day-old fetus was composed of cords of uniform, undifferentiated cells which contained no secretory granules and revealed no peroxidase activity. Peroxidase activity first appeared at the 17th day of gestation in the cisternae of the rough endoplasmic reticulum and nuclear envelope in a few cells. At the 18th day of gestation cells which exhibited reaction products in the rough endoplasmic reticulum and nuclear envelope also contained secretory granules with a strong peroxidase activity. During the last days of gestation the number of peroxidase positive cells, which contained numerous secretory granules, increased. The peroxidase-containing cells are the immediate precursors of the proacinar cells of early postnatal stages. During the same time period, when the peroxidase-containing cells differentiated, a second cell type also differentiated in the cellular cords. The development of this cell type was marked by the appearance of secretory granules stainable with toluidine blue. Through the prenatal development, this cell type revealed no peroxidase activity and was identified with the terminal tubule cell of the newborn. The morphologic and cytochemical findings indicate that terminal tubule cells and proacinar cells are committed cells; the former differentiate toward 2nd order intercalated duct cells and the latter transform to mature acinar cells.

scholarly journals INDUCTION OF ENDOMETRIAL PEROXIDASE SYNTHESIS AND SECRETION BY ESTROGEN AND ESTROGEN ANTAGONIST

1974 ◽  
Vol 62 (2) ◽  
pp. 449-459 ◽  
Author(s):  
Andrew Churg ◽  
Winston A. Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Combined Treatment ◽  
Initial Injection ◽  
Immature Rats ◽  
Glandular Cells ◽  
Endogenous Peroxidase ◽  
Estrogen Antagonist

Synthesis of peroxidase was induced in the uterine epithelium of immature rats by multiple doses over a 24–96-h period of either 17 ß-estradiol, the estrogen-antagonist Parke-Davis CI-628, or a combination of estradiol plus antagonist. Endogenous peroxidase activity first appeared in the cisternae of the rough endoplasmic reticulum of surface epithelial and glandular cells within 24–48 after the initial injection. Uterine peroxidase activity was also visible in the cisternae of the Golgi apparatus, in Golgi-derived secretory granules, and within the uterine and glandular lumen. Some cells of the epithelium produced little or no peroxidase, even after 96 h. Whereas the antagonist appeared to induce synthesis and secretion of peroxidase, neither the antagonist alone nor the combined treatment (estradiol plus antagonist) reproduced the estradiol-mediated growth in organ size and increased lumen diameter.

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