Standardization of Immunohistochemistry Based on Antigen Retrieval Technique for Routine Formalin-fixed Tissue Sections

Shi, Shan; Côté, Richard J.; Chaiwun, Benjaporn; Young, Lillian; Shi, Yan; Hawes, Debra; Chen, Taiying; Taylor, Clive R.

doi:10.1097/00022744-199806000-00006

Cited by 75 publications

(45 citation statements)

References 25 publications

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“…In one early study, five antibodies were tested with results that support the notion that it is possible to achieve Fequalized_ maximal immunostaining levels in FFPE tissue sections fixed in formalin for variable times, as long as 1 month (Shi et al 1998).…”

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confidence: 99%

Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen-retrieval Technique: From Experiments to Hypothesis

Shi

Liu

Taylor

2006

J Histochem Cytochem.

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122

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From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. “The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue”. This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of ∫ (Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as ∫ (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = ∫ (Pffpe)/ ∫ (Pf) × 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf × R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe = Pf. Further studies are designed to test the limitations of the proposed hypothesis.

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confidence: 99%

Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen-retrieval Technique: From Experiments to Hypothesis

Shi

Liu

Taylor

2006

J Histochem Cytochem.

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122

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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%

“…Most of the previous studies of antigen retrieval methods were performed with sections of paraffin-embedded tissues/cells. Depending on the antibodies used, these methods include treatment with various alcohols (to remove paraffi n), different heating temperatures, pressures, osmolality, and chemical composition of the reagents, pH, and different timing of treatments (5)(6)(7)(8)(9)(10). The pH of reagents seems to play a crucial role in antigen retrieval; pH 8 to pH 9 has been proposed as optimal for starting with a new antigen (9).…”

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confidence: 99%

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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“…No entanto, uma das suas principais desvantagens é a formação de "cross--linking" das cadeias de aminoácidos (Eltoum et al 2001, Ramos-Vara 2005, ou seja, promove um "entrelaçamento" das moléculas de proteínas, o que pode alterar, fragmentar ou "esconder" os epítopos e inibir as reações no teste de IHQ (Werner et al 2000, Ramos-Vara 2005. Neste estudo, apesar das reativações antigênicas realizadas para induzir a exposição dos epítopos e otimizar a marcação (Shi et al 1998, Ramos-Vara 2005, não foram observados resultados positivos na maioria dos fragmentos de intestino delgado e de linfonodos mesentéricos com infiltração granulomatosa. Adicionalmente, deve-se considerar que a quantidade e distribuição de Map nas amostras analisadas poderiam estar em níveis não detectáveis pelo protocolo adotado, visto que, a ausência de marcação não significa ausência do antígeno no tecido (Ramos-Vara et al 2008).…”

Section: Teste De Imuno-histoquímicaunclassified

Diagnóstico imuno-histopatológico da paratuberculose subclínica em bovinos no estado do Rio de Janeiro

et al. 2013

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RESUMO.-O diagnóstico precoce e específico da paratuberculose ainda é um desafio. Isto pode estar associado à baixa sensibilidade dos testes laboratoriais e ou à variação da resposta imunológica frente à infecção por Mycobacterium avium subsp. paratuberculosis. The early and specific diagnosis of paratuberculosis remains a challenge due to the low sensitivity of the currently available laboratory tests and also because of variations in the immune response towards infection with Mycobacterium avium subsp. paratuberculosis. Globally this disease causes significant economic losses, primarily in dairy cattle, owing to the chronic nature of the infection. Paratuberculosis has been described in a number of Brazilian states and from a diversity of domestic ruminant species clearly demonstrating that the disease is present in the country and highlighting the requirement for the development of diagnostic techniques for confirmation of infection and for epidemiological analyses. The aim of this study was to characterize the anatomo-histopathological and immunohistochemical findings in the bowel and mesenteric lymph nodes of assymptomatic cattle, derived from paratuberculosis positive herds located in state of Rio de Janeiro, Brazil. Macroscopic examination during necropsy revealed nonspecific changes including reddening of the gut mucosa, increased volumes for the Peyer's patches and mesenteric lymph nodes and in some case dilation and whitening of the mesenteric lymphatic vessel. Histopathology revealed granulomatous infiltration, occasionally with the formation of giant cells in the jejunal and ileal mucosa or sub-mucosa, and/or in the cortical region of the mesenteric lymph nodes, in 32 of the 52 cattle examined. Tissue sections from these animals were subjected to Ziehl-Neelsen staining, but the presence of acid-fast bacilli was not observed. Subsequent analysis, employing genus specific immunohistochemisty for Mycobacterium, revealed areas of immunoreactivity in sections prepared from a total of six animals. The results of this investigation highlighted the value of histopathology and particularly immunohistochemistry as tools for the diagnosis of subclinical paratuberculosis.

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Standardization of Immunohistochemistry Based on Antigen Retrieval Technique for Routine Formalin-fixed Tissue Sections

Cited by 75 publications

References 25 publications

Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen-retrieval Technique: From Experiments to Hypothesis

Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen-retrieval Technique: From Experiments to Hypothesis

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

Diagnóstico imuno-histopatológico da paratuberculose subclínica em bovinos no estado do Rio de Janeiro

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