Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-Nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus.
AuthorsZehbe I, Hacker GW, Su H, Hauser-Kronberger C, Hainfeld JF, Tubbs R
JournalAm J Pathol
PubMed ID9137082
The usefulness of standard in situ hybridization for viral nucleic acid detection is occasionally limited by its sensitivity limit of 10 to 50 copies per cell. A modified version of the recently described signal amplification method, catalyzed reporter deposition (CARD), and its application to formalin-fixed cells and tissue sections is ... More
Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.
AuthorsSun XJ, Tolbert LP, Hildebrand JG
JournalJ Histochem Cytochem
PubMed ID7868862
'Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. ... More
New frontiers in gold labeling.
AuthorsHainfeld JF, Powell RD
JournalJ Histochem Cytochem
PubMed ID10727288
'Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab'' antibody fragments, making small and stable ... More
Highly sensitive DNA, RNA and antigen detection methods: streptavidin-Nanogold-silver staining.
AuthorsHauser-Kronberger C
JournalCell Vis
PubMed ID9660736
Structural studies of the detergent-solubilized and vesicle-reconstituted insulin receptor.
AuthorsWoldin CN, Hing FS, Lee J, Pilch PF, Shipley GG
JournalJ Biol Chem
PubMed ID10574975
'Insulin binding to the insulin receptor initiates a cascade of cellular events that are responsible for regulating cell metabolism, proliferation, and growth. We have investigated the structure of the purified, functionally active, human insulin receptor using negative stain and cryo-electron microscopy. Visualization of the detergent-solubilized and vesicle-reconstituted receptor shows the ... More
The efficacy of in situ PCR, CARD and nanogold systems for gene detection.
AuthorsNuovo GJ
JournalAm J Pathol
PubMed ID9327752
Labeling with nanogold and undecagold: techniques and results.
AuthorsHainfeld JF
JournalScanning Microsc Suppl
PubMed ID9601549
A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and ... More
Sequence-specific molecular lithography on single DNA molecules.
AuthorsKeren K, Krueger M, Gilad R, Ben-Yoseph G, Sivan U, Braun E
JournalScience
PubMed ID12098693
Recent advances in the realization of individual molecular-scale electronic devices emphasize the need for novel tools and concepts capable of assembling such devices into large-scale functional circuits. We demonstrated sequence-specific molecular lithography on substrate DNA molecules by harnessing homologous recombination by RecA protein. In a sequence-specific manner, we patterned the ... More
Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement.
AuthorsLiang RQ, Tan CY, Ruan KC
JournalJ Immunol Methods
PubMed ID14980430
This work presents a method for analyzing protein microarrays using a colorimetric nanogold probe coupled with silver enhancement (gold-silver detection). In this method, the gold nanoparticles were introduced to the microarray by the specific binding of the gold-conjugated antibodies or streptavidins and then coupled with silver enhancement to produce black ... More
High performance Nanogold-silver in situ hybridisation.
AuthorsHacker GW
JournalEur J Histochem
PubMed ID9728288
Conventional in situ hybridisation (ISH) usually requires the presence of at least 10-50 copies of the nucleic acid sequence in question per cell. In situ PCR has been proposed as an alternative method, which may yield single-copy sensitivity, but shows a relatively high rate of false-negative or even false-positive reactions. ... More
Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.
AuthorsWeipoltshammer K, Schöfer C, Almeder M, Wachtler F
JournalHistochem Cell Biol
PubMed ID11201611
Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In ... More
Colorimetric silver detection of DNA microarrays.
AuthorsAlexandre I, Hamels S, Dufour S, Collet J, Zammatteo N, De Longueville F, Gala JL, Remacle J
JournalAnal Biochem
PubMed ID11476538
Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method. Microarrays are ... More
Direct comparison of the sensitivity of enzyme histochemical and immunohistochemical methods: cathepsin B expression in human colorectal mucosa.
AuthorsBleeker FE, Hazen LG, Köhler A, Van Noorden CJ
JournalActa Histochem
PubMed ID10990063
Immunohistochemical localization of the proteinase cathepsin B has been compared directly with localization of cathepsin B activity with a catalytic (enzyme) histochemical method. The 2 approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is active or not whereas the catalytic method ... More
Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy.
AuthorsPowell RD, Halsey CM, Hainfeld JF
JournalMicrosc Res Tech
PubMed ID9712158
Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, ... More
Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes.
AuthorsThompson WF, Beven AF, Wells B, Shaw PJ
JournalPlant J
PubMed ID9351243
Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and ... More
Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.
AuthorsMalecki M
JournalScanning Microsc Suppl
PubMed ID9601525
The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured ... More
Ultrastructural localization of beta-actin and amphoterin mRNA in cultured cells: application of tyramide signal amplification and comparison of detection methods.
AuthorsPunnonen EL, Fages C, Wartiovaara J, Rauvala H
JournalJ Histochem Cytochem
PubMed ID9857217
We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding beta-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold ... More
Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR.
AuthorsJi M, Hou P, Li S, He N, Lu Z
JournalClin Chim Acta
PubMed ID15026275
BACKGROUND: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the ... More
Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility.
AuthorsLe PU, Benlimame N, Lagana A, Raz A, Nabi IR
JournalJ Cell Sci
PubMed ID10954421
Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin ... More
Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers.
AuthorsHumbel BM, de Jong MD, Müller WH, Verkleij AJ
JournalMicrosc Res Tech
PubMed ID9712162
For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, ... More
Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization.
We compared three in situ hybridization (ISH) methods for their applicability and sensitivity in detecting human papillomavirus (HPV) in 61 cases (1 Grade 1, 18 Grade 2, 42 Grade 3) of routinely processed squamous cell cervical carcinoma. A commercially available biotinylated probe for HPV-16/18 was applied to serial sections and ... More
Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.
AuthorsKöhler A, Lauritzen B, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID10858270
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 ... More
Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma.
AuthorsGraf AH, Cheung AL, Hauser-Kornberger C, Dandachi N, Tubbs RR, Dietze O, Hacker GW
JournalAppl Immunohistochem Mol Morphol
PubMed ID11127922
Three different in situ hybridization (ISH) methods were compared for their clinical relevance and suitability in detecting human papillomavirus (HPV) 16/18 in 55 cases of squamous cell carcinoma (SCC) of the uterine cervix. After the initial biopsy, surgery, and/or radiation therapy, patients were followed for 5 to 8 years. A ... More
Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.
Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic ... More