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Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei
Uppsala University, Interfaculty Units, Centre for Image Analysis. Teknisk-naturvetenskapliga vetenskapsområdet, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
Teknisk-naturvetenskapliga vetenskapsområdet, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
2002 (English)In: Cytometry, ISSN 0196-4763, Vol. 47, no 1, 32-41 p.Article in journal (Refereed) Published
Abstract [en]

Background

Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens.

Methods

Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data.

Results

The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample.

Conclusions

The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.

Place, publisher, year, edition, pages
2002. Vol. 47, no 1, 32-41 p.
National Category
Computer Vision and Robotics (Autonomous Systems)
Identifiers
URN: urn:nbn:se:uu:diva-42510OAI: oai:DiVA.org:uu-42510DiVA: diva2:70412
Available from: 2005-08-25 Created: 2005-08-25 Last updated: 2017-02-08

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http://www3.interscience.wiley.com/cgi-bin/abstract/106592262/ABSTRACT

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Wählby, CarolinaBengtsson, Ewert

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