The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique

Mepham, B. L.; Frater, W.; Mitchell, Barry

doi:10.1007/bf01005033

Cited by 298 publications

(58 citation statements)

References 5 publications

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“…Treatment of sections with trypsin (Curran & Gregory, 1978;Mepham et al, 1979) was found to be necessary to achieve maximal staining for all three proteins. In non-trypsinized sections staining for fibronectin was weak except in the smooth muscle underlying the submucosa (Figure la, c), and there was essentially no staining for laminin (Figure 2a).…”

Section: Resultsmentioning

confidence: 99%

Laminin and fibronectin in rectal adenocarcinoma: Relationship to tumour grade, stage and metastasis

Förster¹,

Talbot²,

Critchley³

1984

Br J Cancer

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Summary Using an immunoperoxidase procedure, we have examined the distribution of laminin and fibronectin in normal human large intestinal mucosa and in 50 cases of rectal adenocarcinoma for which extensive clinical follow up was available. In normal tissue, laminin staining was largely restricted to basement membranes, including that underlying the epithelial cells, whereas fibronectin was found in both basement membranes and surrounding connective tissue.In rectal carcinomas, basement membrane-like staining for laminin associated with tumour cells was found in only 27 out of the 50 cases studied. Statistical analysis showed that the presence of laminin-containing basement membranes was correlated with low histological grade (well-differentiated tumours), but not with stage (progression through the bowel wall and the development of lymph node metastases) and, in a highly significant way, with a reduced incidence of distant metastases and increased patient survival.Although fibronectin was found in tumour cell basement membranes where these were present, it was also found in the stroma of all 50 tumours. There was no apparent correlation between the presence of stromal fibronectin and grade, stage or development of metastases.

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Section: Resultsmentioning

confidence: 99%

Laminin and fibronectin in rectal adenocarcinoma: Relationship to tumour grade, stage and metastasis

Förster¹,

Talbot²,

Critchley³

1984

Br J Cancer

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“…Among these, FA treatment is the standard method mainly used for Aβ IHC of FFPE brain tissue sections, although it was originally developed for the immunoreactive enhancement of cerebral amyloids (prion protein and Aβ) and systemic amyloids (amyloid A and prealbumin) (Kitamoto et al 1987). The pretreatment of protein digestion with an enzyme such as trypsin had been used for IHC but only in a limited application (Battifora and Kopinski 1986;Huang et al 1976;Mepham et al 1979). In 1991, the advent of the heating AR method was a breakthrough in the field of IHC.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Kai

Shin

Ogino

et al. 2012

J Histochem Cytochem.

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Senile plaques, extracellular deposits of amyloid β peptide (Aβ), are one of the pathological hallmarks of Alzheimer disease (AD). As the standard immunohistochemical detection method for Aβ deposits, anti-Aβ immunohistochemistry combined with antigen retrieval (AR) by formic acid (FA) has been generally used. Here, we present a more efficient AR for Aβ antigen. On brain sections of AD and its mouse model, a double combination of either autoclave heating in EDTA buffer or digestion with proteinase K plus FA treatment reinforced Aβ immunoreactivity. A further triple combination of digestion with proteinase K (P), autoclave heating in EDTA buffer (A), and FA treatment (F), when employed in this order, gave a more enhanced immunoreactivity. Our PAF method prominently visualized various forms of Aβ deposits in AD that have not been clearly detected previously and revealed numerous minute-sized plaques both in AD and the mouse model. Quantification of Aβ loads showed that the AR effect by the PAF method was 1.86-fold (in the aged human brain) and 4.64-fold (in the mouse brain) higher than that by the FA method. Thus, the PAF method could have the potential to be the most sensitive tool so far to study Aβ pathology in AD and its mouse model.

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“…Between the absolute and 95% ethanol washes, samples were incubated for 10 min in a 0.3% H 2 O 2 (v/v) methanol solution to quench endogenous peroxidase activity. Antigen retrieval methods employing enzymatic digestion (0.05-0.25% trypsin; 0.1% pepsin at 37C) and microwave/steamer heating (10 mM citrate buffer, pH 6.0) were tested to optimize SCCA1 and SCCA2 immunostaining (Mepham et al 1979;Shi et al 1993). A 10-min incubation with 1 ϫ trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA.4Na; Gibco BRL, Grand Island, NY) at 37C enhanced SCCA2 staining, whereas neither procedure improved SCCA1 staining.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Co-expression of the Squamous Cell Carcinoma Antigens 1 and 2 in Normal Adult Human Tissues and Squamous Cell Carcinomas

Çataltepe

Gornstein

Schick

et al. 2000

J Histochem Cytochem.

140

120

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SUMMARY Squamous cell carcinoma antigen (SCCA) serves as a serological marker for advanced squamous cell carcinomas (SCCs) and as an indicator of therapeutic response. Recent molecular studies show that the SCCA is transcribed by two almost identical tandemly arrayed genes, SCCA1 and SCCA2. These genes are members of the high molecular weight serine proteinase inhibitor (serpin) superfamily. Although SCCA1 and SCCA2 are 92% identical at the amino acid level, they have distinct biochemical properties. Paradoxically, SCCA1 is an inhibitor of papain-like cysteine proteinases, such as cathepsins L, S, and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, cathepsin G, and mast cell chymase. Using a new set of discriminatory monoclonal antibodies (MAbs) and polymerase chain reaction (PCR) assay, we showed that SCCA1 and SCCA2 were co-expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassall's corpuscles of the thymus, and some areas of the skin. SCCA1 and SCCA2 also were detected in the pseudo-stratified columnar epithelium of the conducting airways. Examination of squamous cell carcinomas of the lung and head and neck showed that SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. Moreover, there was no differential expression between these SCCA "isoforms" in normal or malignant tissues. In contrast to previous studies, these data indicated that the expression of SCCA1 and SCCA2 was not restricted to the squamous epithelium and that these serpins may coordinately regulate cysteine and serine proteinase activity in both normal and transformed tissues.

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The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique

Cited by 298 publications

References 5 publications

Laminin and fibronectin in rectal adenocarcinoma: Relationship to tumour grade, stage and metastasis

Laminin and fibronectin in rectal adenocarcinoma: Relationship to tumour grade, stage and metastasis

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Co-expression of the Squamous Cell Carcinoma Antigens 1 and 2 in Normal Adult Human Tissues and Squamous Cell Carcinomas

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