The use of the peroxidase reaction to obliterate staining of eosinophils by fluorescein-labelled conjugates

Kingston, David G. I.; Pearson, J R

doi:10.1016/0022-1759(81)90346-x

Cited by 12 publications

(7 citation statements)

References 19 publications

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“…Eosinophil autofluorescence was quenched using a modified version of the procedure described by Kingston and Pearson. 28 Noneosinophil-derived endogenous peroxidase activity was quenched for 30 minutes with 1% H 2 O 2 in pH 7.4 PBS containing 0.5% Tween 80. Sections were then washed with PBS and incubated for 3 minutes with 3,3Јdiaminobenzidine substrate.…”

Section: Fixation and 33ј-diaminobenzidine Pretreatment Of Frozen Sementioning

confidence: 99%

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Brown¹,

Knight²,

Pemberton³

et al. 2004

The American Journal of Pathology

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Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.

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Section: Fixation and 33ј-diaminobenzidine Pretreatment Of Frozen Sementioning

confidence: 99%

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Brown¹,

Knight²,

Pemberton³

et al. 2004

The American Journal of Pathology

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“…frozen bovine intestinal tissue sections was most likely due to eosinophils (FAD and NADPH), lipofuscin, or a combination of both. The masking of resident autofluorescent components of tissue has previously been performed using both DAB 18 and SBB. 8,15,16 In the current study, the MFI of tissue treated with DAB was higher when compared with tissue treated with SBB.…”

Section: Discussionmentioning

confidence: 99%

Autofluorescence and Nonspecific Immunofluorescent Labeling in Frozen Bovine Intestinal Tissue Sections: Solutions for Multicolor Immunofluorescence Experiments

Jenvey

Stabel

2017

J Histochem Cytochem.

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Autofluorescent compounds present in intestinal tissue often hinder the ability to utilize multiple, spectrally different, fluorophores. In addition, fixatives and blocking solutions may contribute to background autofluorescence or nonspecific immunofluorescent labeling. During immunofluorescence protocol development, autofluorescent pigments were observed in frozen bovine mid-ileal intestinal tissue sections. Coagulant fixatives, normal serum blocking, histochemical stains Sudan Black B (SBB) and 3,3'-diaminobenzidine (DAB), and spectral separation using imaging software were compared for their ability to reduce autofluorescence, as well as their effect on immunofluorescent labeling. Fluorescent pigments of frozen bovine mid-ileal intestinal tissue sections, most likely caused by eosinophils and lipofuscin, were masked successfully with a combination of DAB and SBB. Little to no statistical differences were observed for all other methods investigated; however, tissue fixed with 1:1 acetone methanol and 10% horse serum diluted in 0.05 M Tris buffer demonstrated lower mean fluorescence intensities. Spectral separation of specific immunofluorescent labeling from background autofluorescence is a simple method for removing unwanted fluorescence; however, successful separation is dependent on tissue and labeling quality.

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“…Upper jejunal biopsy specimens were obtained with a paediatric Crosby capsule. They were divided into at least two portions, which were fixed immediately, one for routine histology and one for immunofluorescence (15). Assessment of the villous morphology, normal or partial villous atrophy, was made as previously described (29).…”

Section: Methodsmentioning

confidence: 99%

“…The anti-IgE was absorbed with K and h light chains before use to eliminate nonspecific immunoglobulin staining (15,16).…”

Section: Methodsmentioning

confidence: 99%

“…Staining for specific antibody with conjugated P L G and BSA was carried out similarly, but after a 15-min wash in PBS, sections were treated with DAB and H 2 0 2 to mask nonspecific fluorescent staining of eosinophils (15) [see also (39)]. Controls were included of similar sections pretreated with unlabeled antigen and sections from control animals where appropriate.…”

Section: Methodsmentioning

confidence: 99%

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Antibody Production to Milk Proteins in the Jejunal Mucosa of Children with Cow's Milk Protein Intolerance

Pearson¹,

Kingston²,

Shiner³

1983

Pediatr Res

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Summary A direct immunofluorescent technique with fluorescein-labeledantigens was used to examine the jejunal mucosa for specific antibody production in 23 children aged 2-25 months referred for diagnostic biopsy. Plasma cells containing antibodies to p-lactoglobulin (PLG) or bovine serum albumin (BSA) were present in I1 of 16 biopsies from patients with cow's milk protein intolerance (CMPI) but only one of eight controls, all on a diet containing cow's milk. Antibody-containing cells (ACC) constituted approximately 0.5-5% of the total (IgA + IgM) plasma cell population. In children with CMPI there may be gastrointestinal symptoms only, or these may be combined with other allergic manifestations such as eczema or asthma. In many CMPI children diagnostic tests for allergy-skin prick tests or RAST-are negative and the serum IgE is not raised (7, 11,20). This is especially so in those children with only gastrointestinal symptoms. But challenge with cow's milk often produces profound changes in the small intestinal mucosa, including villous atrophy of varying degree (9,10,12,34) and increased numbers of eosinophils (41) intra-epithelial lymphocytes (40) and plasma cells (10,21,33,34). It has not been established whether the increase in plasma cells is just part of a generalized, non-specific response of the immune system to antigenic stimulation (23, 37) or whether it represents a specific response to milk-protein antigens at the point of entry.In order to study the mucosal immune response to bovine milk proteins we have developed a simple direct immunofluorescent technique for the presence of plasma cells containing specific antibody to two bovine milk proteins, PLG and BSA. Brief, preliminary reports of this work have been published (16,27). MATERIALS AND METHODSPatients. Twenty-three children, aged 2-25 months, had been routinely referred to us for diagnostic jejunal biopsy. Informed consent to carry out this procedure was obtained from the parents.In 15 children a diagnosis of CMPI was made, based on a history of vomiting, diarrhoea, and weight loss (or exacerbation of eczema in atopic children) when on CM and improvement when on a CM-free diet. Details of presenting symptoms, dietary history, and results of challenges are given in Table 1. Four of these fifteen patients had additional allergies: to eggs (Nos. 6, 13, and 15), chicken, fish, and antibiotics (No. 11). Five of the fifteen had atopic symptoms such as eczema (Nos. 2, 8 and 13), asthma (NO. 13), and urticaria (No. 6). Five patients (Nos. 4, 5, 9, 10 and 11) were challenged with CM at the time of investigation, and had pre-and postchallenge biopsies as previously described (34); patient No. 4 had a second challenge at age 18 months. In this group a total of 23 biopsies were performed, 16 while the patient was taking CM and 7 while the patient was on a CM-free diet.Eight children whose symptoms could not be related to consumption of CM served as the control group. (Table 2). All were receiving CM at the time of jejunal biopsy.Biopsy specimens. Upper jej...

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The use of the peroxidase reaction to obliterate staining of eosinophils by fluorescein-labelled conjugates

Cited by 12 publications

References 19 publications

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Autofluorescence and Nonspecific Immunofluorescent Labeling in Frozen Bovine Intestinal Tissue Sections: Solutions for Multicolor Immunofluorescence Experiments

Antibody Production to Milk Proteins in the Jejunal Mucosa of Children with Cow's Milk Protein Intolerance

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