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Immunohistochemical Staining
Immunohistochemistry (IHC) is a technique for identifying cellular
or tissue constituents (antigens)
by means of antigen-antibody interactions, the site of antibody binding being
identified
either by direct labelling of the antibody, or by use of a secondary labelling
method.
In Situ Hybridization (ISH) techniques allow the demonstration of specific nucleic
acid sequences (genes) in their cellular environment.
Above: Human Papillomavirus DNA demonstrated by In Situ
Hybridisation (pink)
in epithelial cells identified by indirect immunofluorescence using antibody
against cytokeratin (green)
To provide greater awareness of how easy it is for scientists to label primary antibodies within their own laboratories; Innova Biosciences (Cambridge UK) has launched a new marketing campaign - Go Direct! Rather than struggling with secondary reagents, scientists with the help of Lightning-Link™ are now able to label an antibody directly with minimal hands on time, less than 30 seconds.
Go Direct!
Direct labeling of antibodies greatly simplifies immunodetection techniques. Without the problems of crossover and/or non-specific binding from secondary reagents it is far easier to obtain high quality results in techniques such as flow cytometry, ELISA, Western blotting and immunohistochemistry. The Lightning-Link™ range, the World’s easiest to use DIY antibody labeling kits, consists of over 40 different labels including enzymes, fluorescent proteins/dyes, tandems, biotin and streptavidin -
The Go Direct! campaign will run throughout 2010 with the objective being to increase awareness of how easy it is to directly label antibodies and in doing so, accelerate pioneering science whilst simultaneously saving both money and time!
Seeing is believing! For a timed demonstration video of the Lightning-Link™ antibody labeling process, please visit - Video
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Posted by admin on Monday, January 18 @ 18:43:48 CET (203 reads) (Read More... | 2168 bytes more | Score: 0)
PLA™ Proximity Ligation Assay technology
News in Immunohistochemistry
A Revolution in the Detection of Proteins
Duolink™ enables researchers to study signalling pathways in native cells and tissue in revolutionizing ways. Based on Olink Bioscience’s proprietary in situ Proximity Ligation Assay (in situ PLA), Duolink™ makes it possible to visualize, localize, and quantify individual protein interactions and modifications using standard fluorescence microscopy. This is the first technology that can readily detect and localize protein interactions without the need of tedious genetic engineering to tag and over-express proteins. Thereby, endogenous protein interactions in cells and tissue can be visualized using your regular immuno-staining antibodies combined with the generic Duolink™ kit.
Each spot makes the difference
The unique abiltity of in situ PLA™ to detect protein modifications, interactions, and their location in tissue samples offers exciting new opportunities in biomarker research. By providing the element of dual recognition to in situ analyses, pathology can expand into a new arena of analytes, beyond measuring the abundance of just a single protein.
PLA™, a proximity ligation assay technology, is capable of detecting single protein events such as protein interactions (e.g. protein dimerization) and modifications (e.g. protein phosphorylation).
The Duolink® reagent series, based on PLA, offers an unprecedented level of sensitivity and specificity for protein detection in fixed cells and tissues. The principle of the technology is based on two unique bi-functional probes called PLA probes. Each PLA probe consists of an antibody attached to a unique synthetic oligonucleotide, which acts as a reporter.
The assay provides exact spatial information on the location of the events and an objective means of quantifying the events.
In principle, with appropriate antibodies, in situ PLA can detect any antigen with proximate epitopes at the single molecule level.
Posted by admin on Monday, December 21 @ 10:14:42 CET (957 reads) (Read More... | 8559 bytes more | Score: 5)
Diagnostic Immunohistochemistry
Diagnostic Immunohistochemistry: Theranostic and Genomic Applications, Expert Consult: Online and Print
By David J. Dabbs MD
Diagnostic Immunohistochemistry presents the latest information and most reliable guidance on immunohistological diagnoses in surgical pathology. David J. Dabbs, MD and other leading experts bring you state-of-the-art coverage on genomic and theranostic applications, molecular anatomic pathology, immunocytology, Non-Hodgkin's lymphoma, and more. Additional features such as tables discussing antibody specifications, differential diagnosis boxes, ancillary anatomic molecular diagnostics, and full-color histological images ensure user-friendly coverage that makes key information easy to find and apply. The fully searchable text is also available online at expertconsult.com, along with a downloadable image bank and access to Path Consult. This concise and complete resource is today's indispensable guide to the effective use of immunohistochemical diagnosis.
Posted by admin on Thursday, October 22 @ 11:11:53 CEST (565 reads) (Read More... | 4020 bytes more | Score: 5)
Human Antibody Initiative
The mission of the Human Antibody Initiative (HAI) aims to promote and facilitate the use of antibodies for proteomics research. The initiative consists of two separate activities; (1) the generation of a catalogue of validated antibodies from many different sources and (2) a protein atlas for the expression and localization of human proteins in normal and disease tissue. The two separate activities have as their primary deliverables to generate databases with free public accessibility. The Antibody Resource database (www.antibodypedia.org) is aimed to produce a comprehensive catalogue of validated antibodies towards human proteins. This initiative depends on input from a large number of academic groups and commercial companies. The Protein Atlas initiative (www.proteinatlas.org) is aimed to provide comprehensive and annotated database of high-resolution images showing tissue profiles in normal and cancer tissues. Both databases will be open to the public without restriction (no passwords).
The Antibodypedia portal is a database developed within the 6th framework EU program ProteomeBinders and the project is part of the HUPO antibody initiative (HAI). This pilot portal provides a web-based submission format to allow any antibody provider or user to submit data about antibodies with validation scores for various applications. It is only possible to submit antibodies available to the scientific community. The database relies on validation scores, submitted by the antibody provider or user, based on a standard set of validation criteria, but it is important to point out that the validation is subjective in nature. It is thus mandatory to submit the primary data for applications with supportive or uncertain validation score, usually in the form of an image with text annotation, to allow the community to review the data behind the validation score. Users are also allowed to send in comments to the portal about the use of a particular antibody and in this manner both positive and negative results from a particular antibody can be shared among the scientific community. No curation of the data is done in the first phase, but the plan is to incorporate an independent curation step to ensure that the formal submission rules have been followed.
Vector Laboratories Inc is offering a comprehensive, illustrated guide to multiple antigen labeling for immunohistochemistry and immunofluorescent staining in tissues or cells.
A valuable resource for those considering localizing two or more antigens in the same tissue section. Protocols for chromogenic as well as fluorescent detection are accompanied by schematic illustrations of the procedures. A chart of all pair-wise combinations of substrates is included along with photographic examples. Practical tips regarding order of substrates, blocking, and proper controls are also provided.
Download a copy here (Vector Laboratories, pdf 8.2MB)
Posted by admin on Friday, August 28 @ 09:44:40 CEST (786 reads) (Read More... | Score: 0)
Modern Immunohistochemistry
Modern Immunohistochemistry (Cambridge Illustrated Surgical Pathology)
Diagnostic pathology is an inherently flawed science due to the many possible interpretations of various tissues. However, the application of immunohistochemical stains as a diagnostic tool has been widely used since the 1990s as an extremely effective ancillary technique that removes much subjectivity from the practice. In fact, immunohistochemistry has supplanted simple morphologic evaluation as the definitive diagnostic method for a wide array of tumor types. This book offers a new and modern atlas-based resource for this science. Every anatomic region is covered in detail, and major diseases contain side-by-side examples of other ancillary staining techniques for comparison. The text is geared toward both the resident and practitioner of anatomic pathology and is supplemented with histograms, algorithms, and guides to the application and interpretation of uncommon antigens and immunostains. Not only is the book illustrated with more than 600 high-quality photomicrographs, but a companion CD-ROM of all images in downloadable format is included.
Posted by admin on Friday, June 19 @ 21:13:45 CEST (984 reads) (Read More... | Score: 0)
Immunohistochemistry - NeuroMabs
The mission of the NeuroMab facility is to provide a unique neuroscience-based approach to generating mouse monoclonal antibodies optimized for use in mammalian brain (NeuroMabs). NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mAbs that are optimized for use in the brain for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks.
The initial goal of the facility for the next 5 years is to generate a library of novel NeuroMabs against 250-500 neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatant or purified immunoglobulin by Antibodies Inc.
Posted by admin on Monday, March 09 @ 08:00:00 CET (1221 reads) (Read More... | Score: 0)
Video Protocols
The Journal of Visualized Experiments (JoVE)
The Journal of Visualized Experiments (JoVE) has announced that its online video protocols will be indexed in the popular US National Library of Medicine repositories MEDLINE and PubMed.
Founder and chief executive Moshe Pritsker views the MEDLINE–PubMed listing as a sign that the scientific community has accepted video-based publications. "It was a very important decision for us, and for scientific publishing," he says.
Since JoVE was founded in 2006 with support from an angel investor, the journal has published more than 200 videos, most produced by professional videographers. It aims to improve the reproducibility of scientific results by using videos to clarify subtle experimental details. The journal was itself an experiment in video publishing and remains the only video-based scientific journal.
Immunocytochemistry: Human Neural Stem Cells
Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 μl of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.
A Revolution in the Detection of Proteins
Diagnostic Immunohistochemistry:
Vector Laboratories Inc is offering a comprehensive, illustrated guide to multiple antigen labeling for immunohistochemistry and immunofluorescent staining in tissues or cells.
Diagnostic pathology is an inherently flawed science due to the many possible interpretations of various tissues. However, the application of immunohistochemical stains as a diagnostic tool has been widely used since the 1990s as an extremely effective ancillary technique that removes much subjectivity from the practice. In fact, immunohistochemistry has supplanted simple morphologic evaluation as the definitive diagnostic method for a wide array of tumor types. 
The mission of the NeuroMab facility is to provide a unique neuroscience-based approach to generating mouse monoclonal antibodies optimized for use in mammalian brain (NeuroMabs). NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mAbs that are optimized for use in the brain for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. 