Criteria of reliability for light microscopic immunocytochemical staining

Petrusz, Peter; Ordronneau, Paul; Finley, James C. W.

doi:10.1007/bf01006954

Cited by 113 publications

(32 citation statements)

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“…20 In immunohistochemical terms, specificity should be discussed in sets of two criteria, method specificity and antibody specificity. 27 Method specificity is the absence of any unwanted staining, but antibody specificity is more difficult to evaluate. Regarding the use of immunologic tests for the diagnosis of BISD, cross reactions to other bacteria have been observed for monoclonal 1 and polyclonal 28 antibodies, but such reactions have not been described for MAbs 4D3 and 2G5.…”

Section: Discussionmentioning

confidence: 99%

“…7 Specificity in immunohistochemistry is troublesome, especially because antibody specificity is difficult to determine. 27 Using polyclonal antisera, lot variations occur, and cross-reactivity is commonly associated with such sera, also being observed for polyclonal antisera against R. salmoninarum. 28 Cross-reactivity has also been reported for monoclonal antibodies (MAbs) against R. salmoninarum in enzyme-linked immunosorbent assay techniques, l although recently MAbs, 4D3 and 2G5, have been reported to identify 2 different surface protein epitopes unique for R. salmoninarum.…”

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confidence: 99%

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Immunohistochemical Identification of Renibacterium Salmoninarum by Monoclonal Antibodies in Paraffin-Embedded Tissues of Atlantic Salmon (Salmo Salar L.), using Paired Immunoenzyme and Paired Immunofluorescence Techniques

Evensen

Dale

Nilsen³

1994

J VET Diagn Invest

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Abstract. Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate. Antigen detection sensitivity based on an immunoenzymatic alkaline phosphatase method and Fast Red as chromagen and evaluated by an image-analysis system creating a relative grey-level score showed an 8-fold higher detection sensitivity on ethanol-fixed as compared with formalin-fixed specimens. Monoclonal antibodies specific for the outer membrane protein of Renibacterium salmoninarum are well suited for use in immunohistochemical techniques for in situ identification of the bacterium in paraffin-embedded tissue specimens. Serial evaluation of paired stained sections with a confirmatory diagnosis of BKD being dependent on both MAbs giving a positive reaction would increase the specificity of the test. Simultaneous identification of the two epitopes of p57 on the same section was dependent on ethanol-fixed tissue specimens.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Immunohistochemical Identification of Renibacterium Salmoninarum by Monoclonal Antibodies in Paraffin-Embedded Tissues of Atlantic Salmon (Salmo Salar L.), using Paired Immunoenzyme and Paired Immunofluorescence Techniques

Evensen

Dale

Nilsen³

1994

J VET Diagn Invest

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“…Method specificity (25) was tested by a series of increasing dilutions of the primary antiserum, resulting in a gradual decrease and eventual disappearance of the immunostaining. Antiserum specificity was tested by absorption of the anti-CRF serum with synthetic CRF, bovine insulin (Sigma), glucagon (Sigma), synthetic SR-IF-14 (Beckman Bioproducts, Palo Alto, CA), pentagastrin (Bachem Fine Chemicals, Marina Proc.…”

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confidence: 99%

Corticotropin-releasing factor (CRF)-like immunoreactivity in the vertebrate endocrine pancreas.

Petrusz¹,

Merchenthaler²,

Maderdrut³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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The light microscopic immunocytochemical localization of corticotropin-releasing factor (CRF) is described in the endocrine pancreas of several species representing the major classes of vertebrates: fishes (channel catfish, Ictalurus punctatus), amphibians (African clawed toad, Xenopu8 laevis), reptiles (chameleon, Anolis carolinensis), birds (chicken, Gallus domesticus), and several mammals (rat, mouse, cat, rhesus monkey, and man). The CRF-containing cells are scattered over the entire islet tissue in primates and cat, whereas in rat and mouse they are located at the periphery of the islets. In the chicken and catfish, the CRF-containing cells are found in a central location within islets and form larger clusters or cords. Single cells with CRF-like immunoreactivity are interspersed between acinar cells of the exocrine pancreas in all species studied. The CRF cells show a substantial topographical overlap with glucagon cells, but their precise identity and function remain to be determined.Corticotropin-releasing factor (CRF), a 41-amino acid peptide, has been isolated and characterized from extracts of ovine hypothalamus as a potent stimulator of the secretion of corticotropin (ACTH) and 3-endorphin (1-3). Immunocytochemical studies have established that most of the CRF in the hypothalamus is located both in neurons of the paraventricular nucleus and in terminals around portal capillaries of the median eminence (4-7). CRF-like immunoreactivity has been identified in hypophysial portal blood (8). The neural pathways through which CRF reaches the median eminence also have been described (9). Like other neuropeptides, CRF is widely distributed in extrahypothalamic areas of the brain, determined by both radioimmunoassay (10) and immunocytochemistry (11)(12)(13). In addition to stimulating the release of ACTH and /8-endorphin, CRF has a broad range of pharmacological effects, which include changes in behavior, heart rate, blood pressure, and in blood concentrations of epinephrine, norepinephrine, glucagon, and glucose (14-18). Thus, CRF appears to be an important regulatory peptide mediating stress-related physiological responses and predictably possessing other, hitherto unsuspected, hormone-or neurotransmitter-like activities. On the basis of current hypotheses concerning the common evolutionary origin of hormones and neurotransmitters (19)(20)(21) (Altamont, NY). One African clawed toad (Xenopus laevis) and pancreatic tissue from two channel catfish (Ictalurus punctatus) were gifts from L. J. Haverkamp (Neurobiology Program, University of North Carolina) and J. E. Brinn (Department of Anatomy, East Carolina University), respectively. Three lizards (Anolis carolinensis) were purchased from Carolina Biological Supply (Burlington, NC). Pancreatic tissue from two adult cats and two adult rhesus monkeys (Macaca mulatta) also was studied. Human pancreas (courtesy of E. K. MacRae and F. D. Dalldorf) was from a 1-year-old infant who died in an accident.Preparation of Tissues. Rat, mouse, cat, monkey, chicken, ...

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“…The main parameters which should be fulfilled in a satisfactory manner have been classified by PETRUSZ et al (1980PETRUSZ et al ( , 1983 in the following terms: standardization, reproducibility, precision, accuracy, and efficiency. In accord with these parameters we have tested immunohistochemical methods including immunofluorescence, the labelled and non-labelled antibody enzyme technique, the avidin-biotin-complex (ABC) method, and immunogold procedures (LARSSON, 1981;MEY, 1983;NOORDEN and POLAK, 1983;STERNBERGER, 1986) in semithin sections.…”

Section: Immunohistochemical Techniquesmentioning

confidence: 99%

“…For immunostaining using the PAP method we strongly recommend incubation of the sections with highly diluted antisera (first layers) and an incubation time of 24 hr (see also BIGBEE et al, 1977;GRUBE,1980a;PETRUSZ et al, 1980;STERNBERGER, 1986). In addition to economical advantages, a high dilution of the first antiserum minimizes the pitfalls caused by the non-specific binding mechanisms of antibodies to tissue components (BUFFA et al, 1979;GRUBE, 1980a;GRUBE and WEBER,1980;STERNBERGER, 1986).…”

Section: Immunohistochemical Techniquesmentioning

confidence: 99%