Efficient inhibition of endogenous peroxidase without antigen denaturation in immunohistochemistry

Malorny, Ursula; Bildau, Horst; Sorg, Clemens

doi:10.1016/0022-1759(88)90065-8

Cited by 21 publications

(13 citation statements)

References 11 publications

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“…Ear sections were processed for immunoperoxidase staining as previously described [15]. Briefly, 5 pm cryostat sections were ace tone-fixed and blocked with 1% bovine serum albumin (Sigma) in phosphate-buffered saline after inhibiting endogenous peroxidase with sodium azide in 0.3% hydrogen peroxide.…”

Section: Immunohistochemislrymentioning

confidence: 99%

Expression of Intercellular Adhesion Molecule-1 in Murine Allergic Contact Dermatitis

Goebeler¹,

Gutwald²,

Roth³

et al. 1990

Int Arch Allergy Immunol

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Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the interaction of immunocompetent cells in inflammation. It contributes to lymphocyte recruitment, antigen presentation and T-cell activation. Recently, the murine homologue of ICAM-1, originally designated as MALA-2, has been identified. The monoclonal antibody YN1/1.7 for murine ICAM-1 enables the analysis of ICAM-1 expression in murine allergic contact dermatitis, a prototype of cutaneous T-cell-mediated inflammatory response. At 0, 1, 8, 24, 48, 72 h, and 7 and 14 days after challenge with 2,4-dinitrofluoro-1-benzene (DNFB) cryostat sections of ear skin were immunostained for ICAM-1, I-A and mononuclear cells (L3T4, Lyt-2, BM8). In normal skin ICAM-1 labeling was restricted to endothelial cells and dermal dendritic cells/macrophages; keratinocytes (KCs) did not express ICAM-1. The dermal cellular infiltrate increased progressively from 8 to 72 h after DNFB challenge. The majority of infiltrating cells were BM8+ macrophages (75%) and L3T4+ (10%) or Lyt-2 + T cells (10%); maximally 30% of those stained positive for ICAM-1. At 24 h, focal ICAM-1 expression on KCs developed, reached a maximum at 72 h and faded therafter. Migration of T cells into the epidermal layer started at 48 h at sites which had already expressed ICAM-1. Our data provide evidence that expression of ICAM-1 by epidermal cells precedes infiltration of the epidermis by T lymphocytes as shown before in human cutaneous disorders. Thus, a mouse model may be useful to investigate the role of ICAM-1 in inflammation further.

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Section: Immunohistochemislrymentioning

confidence: 99%

Expression of Intercellular Adhesion Molecule-1 in Murine Allergic Contact Dermatitis

Goebeler¹,

Gutwald²,

Roth³

et al. 1990

Int Arch Allergy Immunol

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“…To block the EPX activity, different methods were tested (Straus 1971(Straus , 1972Streefkerk 1972;Vacca et al 1978;Kelly et al 1987;Malorny et al 1988). The method, described by Streefkerk (1972) was found to be the most successful in preserving the tissue structure, altough the EPX blockade was not complete.…”

Section: Blocking the Epx Activitymentioning

confidence: 99%

“…Certain hemopoietic cells are reported to show endogenous peroxidase (EPX) or pseudoperoxidase activity in sections of human lung (Vacca et al 1978), small intestine (Kelly et al 1987), mice lymph nodes (Malorny et al 1988), rat spleen (Streefkerk, 1972), and other organs (Straus 1979). The presence of these cells in different tissues may interfere with histochemical methods, based on the peroxidase-catalyzed color reaction of the diaminobenzidine (DAB) reagent (Sternberger et al 1970;Heyderman 1979;Straus 1979;Elias 1990).…”

Section: Introductionmentioning

confidence: 98%

“…The presence of these cells in different tissues may interfere with histochemical methods, based on the peroxidase-catalyzed color reaction of the diaminobenzidine (DAB) reagent (Sternberger et al 1970;Heyderman 1979;Straus 1979;Elias 1990). A variety of procedures have been reported for the removal of the EPX and pseudoperoxidase activity from tissues (Straus, 1971(Straus, , 1972Streefkerk and Ploeg 1973;Vacca et al 1978;Kelly et al 1987;Malorny et al 1988), however, none of these methods was found to be satisfactory in cryostat sections from the rat gastrointestinal (GI) system, in terms of either the completeness of the EPX blockade or preserving the integrity of the antigens to be investigated. We studied the EPX containing cells (EPX cells) in cryostat sections of the GI tract from four aspects: (1) distribution of EPX cells in the rat GI tract; (2) description of a sufficient and antigen-preserving blocking method to remove the EPX activity from the tissue; (3) identification of the cells responsible for the histochemically demonstrable EPX activity in the rat GI system; and (4) investigation of the distribution of EPX cells in pathological circumstances, i.e., in sections from the stomach of immobilized rats and from ulcerated human stomachs.…”

Section: Introductionmentioning

confidence: 99%

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Identification of endogenous peroxidase-containing cells as eosinophils in the gastrointestinal system

Hunyady

Mezey

Pacák

et al. 1996

Histochem Cell Biol

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Endogenous peroxidase (EPX) activity in certain cells in the gastrointestinal system interferes with immunohistochemical methods based on the horseradish peroxidase-catalyzed substrate deposition. We studied the distribution and characteristics of these cells. We also report an effective and antigen-preserving EPX blocking method, to make possible the evaluation of immunoperoxidase stainings in cryostat sections. The EPX-containing cells (EPX cells) are present in every part of the gastrointestinal tract, predominantly in the tunica propria. We identified them as eosinophil cells in May-Grünwald-Giemsa stained sections. The complete match was confirmed by different fluorescence techniques. Firstly, the EPX cells were labeled by a red fluorochrome-conjugated substrate of peroxidase enzymes, rhodamine-tyramide, whereas the eosinophil cells were labeled by the green fluorochrome, l-hydroxy-3,6,8-pyrenetrisulfonic acid, which is known to label exclusively eosinophilic granules at pH 10. Secondly, all the EPX cells reacted with a monoclonal antibody against the eosinophil peroxidase enzyme. Finally, a set of commercially available leukocyte markers was used to characterize the EPX cells colabeled by fluorochrome-tyramides. Neither macrophages nor mast cells showed EPX activity. Increased numbers and altered distribution were seen in stressed rats and in ulcerated human stomach.

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“…while the other one was fixed in 10% formalin in phosphate-buffered saline and embedded in paraffin. Five-micro meter paraffin or acetone-fixed cryostat sections were processed for immunoperoxidase staining as previously described [18]. Briefly, cn- dogenous peroxidase was inhibited by sodium azide/H ,0, which was followed by blocking with 1% bovine serum albumin (Sigma) in phos phate-buffered saline.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Changes in Phenotypically Distinct Phagocyte Subpopulations during Nonspecific Modulation of Contact Sensitization

Frantzen

Goerdt²,

Goebeler³

et al. 1993

Int Arch Allergy Immunol

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Environmental influences are increasingly recognized as nonspecific modulators triggering and aggravating allergic diseases. To obtain better insight into the pathomechanisms of these nonantigen-specific phenomena, we studied the augmenting and inhibitory effects of croton oil and glucocorticosteroid (GC) on the induction of murine allergic contact dermatitis. Application of the irritant croton oil simultaneously with the sensitization dose of the contact allergen oxazolone strongly amplified the inflammatory reaction (measured as ear swelling) during the effector phase. Immunohistologically, this effect correlated with an increased percentage of MRP8- and MRP14-positive phagocytes in the infiltrate of the early inflammatory reaction 8 h after sensitization with oxazolone plus croton oil as compared to oxazolone alone (62 vs. 27%; p < 0.05). Intravenously administered GC was used as an inhibitor of contact sensitization. The suppressive effect of GC on sensitization was dependent on the time of its application: suppression of the inflammatory reaction during the effector phase was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization. Correspondingly, the percentage of BM8-positive macrophages in the infiltrate of the early inflammatory reaction 8 h after sensitization was differentially decreased depending on the time of GC application: suppression of the percentage of BM8-positive macrophages was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization (17 vs. 25%; base value without GC 35%; p < 0.05). Furthermore, the percentage of BM8-positive macrophages in the inflammatory infiltrate of the effector phase was increased when sensitization had been enhanced by concomitant irritant contact dermatitis (47 vs. 59%; p < 0.05). Thus, nonspecific modulation of contact sensitization is accompanied by significant changes in the composition of the inflammatory infiltrate by phenotypically distinct phagocyte subpopulations suggesting that these cells may play an active role in the pathophysiology of contact sensitivity.

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Efficient inhibition of endogenous peroxidase without antigen denaturation in immunohistochemistry

Cited by 21 publications

References 11 publications

Expression of Intercellular Adhesion Molecule-1 in Murine Allergic Contact Dermatitis

Expression of Intercellular Adhesion Molecule-1 in Murine Allergic Contact Dermatitis

Identification of endogenous peroxidase-containing cells as eosinophils in the gastrointestinal system

Changes in Phenotypically Distinct Phagocyte Subpopulations during Nonspecific Modulation of Contact Sensitization

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