Optimizing immunohistochemistry: antigen retrieval and signal amplification

McNicol, A. M.; Richmond, James

doi:10.1046/j.1365-2559.1998.00342.x

Cited by 73 publications

(50 citation statements)

References 84 publications

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“…Harsh antigen retrieval methods, such as delipidation with alcohols, microwave heating in buffers of different pH, and autoclaving, are usually used to recover cryptic epitopes in sections of formalin-fi xed, paraffi n-embedded tissues (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)12). Such techniques have not regularly been used with cryosections.…”

Section: Discussionmentioning

confidence: 99%

“…However, fi xation with PFA has some drawbacks; it "masks" the antibody binding sites (epitopes) on some antigens, and alters cellular morphology by damaging the cell structure (1). It is not clear how PFA reacts with proteins, except that it cross-links reactive sites within the same proteins and between adjacent proteins via methylene bridges (2). However, not all proteins are masked by PFA at the same rate and intensity.…”

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confidence: 99%

“…For instance, monoclonal antibodies fail to label neuro-fi laments after one day, lymphocyte antigens LN1, LN2, and LN3 have shown reduced staining after three days, while carcinoembryonic antigen stains even after 14 days of tissue fi xation in formalin (3). This problem is more evident for monoclonal then polyclonal antibodies, because polyclonal antibodies can bind to more reactive places on proteins (2). In order to "unmask" cryptic epitopes, one has to apply a series of antigen retrieval techniques for each set of antigen/antibody separately in order to fi nd optimal conditions for the immunocytochemical presentation of an antigen.…”

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confidence: 99%

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Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Brzica

Breljak

Ljubojević

et al. 2009

Archives of Industrial Hygiene and Toxicology

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Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat KidneyTo localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Brzica

Breljak

Ljubojević

et al. 2009

Archives of Industrial Hygiene and Toxicology

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“…Immunohistochemistry was performed based on a standard optimized protocol of McNicol and Richmond (1998) with few modifications as standardized earlier in our previous study . The paraffin-embedded unstained section of myocardial tissues were deparaffinized in the xylene and followed by subsequent rehydration with descending series (100%, 90%, 70%, 50%, 30%) of ethanol.…”

Section: Immunohistochemical Analysismentioning

confidence: 99%

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride

Panneerselvam

Raghunath

2017

Cell Stress and Chaperones

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Acute fluoride (F − ) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F − induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (HspsHsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F − -intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F − for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F − -intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dosedependent fashion in F − -treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F − -induced heart failure.

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“…Moreover, some studies (Kropveld et al, 1999 andBalz et al, 2003) have shown that the analysis of the entire coding region of TP53 (exons 2-11) in head and neck SCC increases the frequency of mutations and yields a greater concordance level with immunohistochemistry results, although many caveats may lead to false-positive or false-negative results (Piffkó et al, 1995;Dowell & Ogden, 1996;McNicol & Richmond, 1998;Stoll et al, 1998;Saunders et al, 1999;Nylander et al and Gonzalez-Moles et al).…”

Section: P53 Immunoexpression In Oral Squamous Cell Carcinomas From Dmentioning

confidence: 99%

p53 Immunoexpression in Oral Squamous Cell Carcinomas from Different Anatomical Sites: A Comparative Study

Sá¹,

Fonseca²,

Cardoso³

et al. 2006

Int. J. Morphol.

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SUMMARY: p53 alterations have been implicated in oral carcinogenesis process. Squamous cell carcinoma is the most common malignancy of the oral cavity, the tongue being the most frequent affected site. The aim of this study was to compare p53 immunoexpression in 43 samples of oral SCC from 5 different anatomical sites: tongue, floor of the mouth, alveolar ridge/gingiva, retromolar region and other regions of the oral cavity. Thirty-three lesions (76,8%) showed positivity for p53 (mean index of 48,37%). No statistically significant differences were found between p53 expression and the different oral sites analyzed. Similarly, no differences were found when comparing tongue lesions with the remaining sites. These results suggest that it might not exist any difference among oral cavity anatomical sites in relation to p53 immunoexpression. Moreover, mechanisms other than p53 alterations in squamous cell carcinomas of the tongue may be of relevance for the higher tumour aggressiveness found in this anatomical site.KEY WORDS: p53 protein; Immunohistochemistry; Oral squamous cell carcinoma (OSCC).

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Optimizing immunohistochemistry: antigen retrieval and signal amplification

Cited by 73 publications

References 84 publications

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride

p53 Immunoexpression in Oral Squamous Cell Carcinomas from Different Anatomical Sites: A Comparative Study

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