Ultrastructural Localization of β-Actin and Amphoterin mRNA in Cultured Cells: Application of Tyramide Signal Amplification and Comparison of Detection Methods

Punnonen, Eeva‐Liisa; Fages, Carole; Wartiovaara, Jorma; Rauvala, Heikki

doi:10.1177/002215549904700111

Cited by 20 publications

(14 citation statements)

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“…The sections were subsequently exposed to preformed biotin-peroxidase/streptavidin complex for 30 minutes and the antigen-antibody complexes were visualized with 0.7 mM diaminobenzidine hydrochloride/0.002% H 2 O 2 in 0.05 M Tris HCl (pH 7.6). To increase the signal, 6 mg/ml ammonium-nickel sulphate was added to the diaminobenzidine–H 2 O 2 solution wherever indicated [23]. To confirm the specificity of immunohistochemical staining, the previously described control stainings were performed [9].…”

Section: Methodsmentioning

confidence: 99%

Hepcidin Is an Antibacterial, Stress-Inducible Peptide of the Biliary System

et al. 2011

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Background/AimsHepcidin (gene name HAMP), an IL-6-inducible acute phase peptide with antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of HAMP synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. While the role of hepcidin in biliary system is unknown, a recent study demonstrated that conditional gp130-knockout mice display diminished hepcidin levels and increased rate of biliary infections.MethodsExpression and localization of HAMP in biliary system was analyzed by real time RT-PCR, in-situ hybridization, immunostaining and –blotting, while prohepcidin levels in human bile were determined by ELISA.ResultsHepcidin was detected in mouse/human gallbladder and bile duct epithelia. Biliary HAMP is stress-inducible, in that it is increased in biliary cell lines upon IL-6 stimulation and in gallbladder mucosa of patients with acute cholecystitis. Hepcidin is also present in the bile and elevated prohepcidin levels were observed in bile of primary sclerosing cholangitis (PSC) patients with concurrent bacterial cholangitis compared to PSC subjects without bacterial infection (median values 22.3 vs. 8.9; p = 0.03). In PSC-cholangitis subjects, bile prohepcidin levels positively correlated with C-reactive protein and bilirubin levels (r = 0.48 and r = 0.71, respectively). In vitro, hepcidin enhanced the antimicrobial capacity of human bile (p<0.05).ConclusionHepcidin is a stress-inducible peptide of the biliary epithelia and a potential marker of biliary stress. In the bile, hepcidin may serve local functions such as protection from bacterial infections.

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Section: Methodsmentioning

confidence: 99%

Hepcidin Is an Antibacterial, Stress-Inducible Peptide of the Biliary System

et al. 2011

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“…However, studies in our laboratory have suggested that localization of amphoterin mRNA to the cell periphery might be a key step in bypassing nuclear transport of amphoterin. Localization of amphoterin mRNA shares similar features with β ‐actin mRNA localization, and these two mRNA particles are often found in cell processes together with ribosomes suggesting local mode of translation [18, 65]. Thus, mechanisms controlling localization of amphoterin mRNA might also be important regulators of secretion of amphoterin.…”

Section: Expression and Secretion Of Amphoterinmentioning

confidence: 99%

Amphoterin as an extracellular regulator of cell motility: from discovery to disease

Huttunen

Rauvala

2004

Journal of Internal Medicine

159

128

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Abstract. Huttunen HJ, Rauvala H (University of Helsinki, Helsinki, Finland). Amphoterin as an extracellular regulator of cell motility: from discovery to disease (Minisymposium). Amphoterin is a ubiquitous and highly conserved protein previously considered solely as a chromatinassociated, nuclear molecule. Amphoterin is released into the extracellular space by various cell types, and plays an important role in the regulation of cell migration, differentiation, tumorigenesis and inflammation. This paper reviews recent research on the mechanistic background underlying the biology of secreted amphoterin, with an emphasis on the role of amphoterin as an autocrine/paracrine regulator of cell migration.

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“…Then sections were incubated with DAB and H 2 O 2 , rinsed, counterstained, and mounted as described in the ABC-DAB method. In some cases, 6 mg/ml ammonium-nickel sulfate was added to the DAB-H 2 O 2 solution as recommended by Punnonen et al (1999). The solution was filtered when cloudy.…”

Section: Avidin-biotin Complex With Diaminobenzidine (Abc-dab)mentioning

confidence: 99%

“…In particular, catalyzed signal amplification (CSA) based on the deposition of labeled tyramide after activation by conversion of H 2 O 2 by peroxidase conjugated with an antibody has greatly increased the sensitivity of IHC and ISH methods (for reviews see Totos et al 1997;Speel 1999;Speel et al 1999). The method was originally developed for ELISA and Western blotting (Bobrow et al 1989(Bobrow et al ,1991(Bobrow et al ,1992 and has been adapted for light microscopy (LM) (Adams 1992;Kerstens et al 1995;Raap et al 1995;Van Gijlswijk et al 1996) and electron microscopy (EM) (Schöfer et al 1997;Bendayan 1997,1999;Punnonen et al 1999). The best detection systems for the CSA technique are at present the diaminobenzidine reaction with nickel enhancement or fluorescence for LM and Nanogold-silver staining for EM (Punnonen et al 1999).…”

mentioning

confidence: 99%

“…The method was originally developed for ELISA and Western blotting (Bobrow et al 1989(Bobrow et al ,1991(Bobrow et al ,1992 and has been adapted for light microscopy (LM) (Adams 1992;Kerstens et al 1995;Raap et al 1995;Van Gijlswijk et al 1996) and electron microscopy (EM) (Schöfer et al 1997;Bendayan 1997,1999;Punnonen et al 1999). The best detection systems for the CSA technique are at present the diaminobenzidine reaction with nickel enhancement or fluorescence for LM and Nanogold-silver staining for EM (Punnonen et al 1999). A great advantage of the CSA technique is that antigens can be detected easily in fixed material that is embedded in paraffin or plastic (Von Wasielewski et al 1997).…”

mentioning

confidence: 99%

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Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold-Silver Staining

Köhler

Lauritzen

Noorden

2000

J Histochem Cytochem.

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(CJFVN) S U M M A R Y Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 g/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.

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Ultrastructural Localization of β-Actin and Amphoterin mRNA in Cultured Cells: Application of Tyramide Signal Amplification and Comparison of Detection Methods

Cited by 20 publications

References 61 publications

Hepcidin Is an Antibacterial, Stress-Inducible Peptide of the Biliary System

Hepcidin Is an Antibacterial, Stress-Inducible Peptide of the Biliary System

Amphoterin as an extracellular regulator of cell motility: from discovery to disease

Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold-Silver Staining

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