| A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy. | |
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MedLine Citation:
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PMID: 17622969 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The aim of this study was to develop a procedure to remove the TO-PRO-3 fluorescent dye from tissue sections and restain with TO-PRO-3, still allowing calculation of DNA content and distribution by confocal laser scanning microscopy (CLSM). This would allow repeated measurements on the same tissue sections and prevents loss of tissue material from valuable clinical samples. Thick sections (14 microm) were cut from a paraffin block of adrenal tissue and stained using TO-PRO-3. Image stacks were acquired by CLSM. Thereafter, three destaining approaches were tested based on incubation, at different temperatures and durations, in the medium that is normally used to dissolve TO-PRO-3. The same areas were imaged again to measure residual fluorescence and were subsequently restained and imaged again. The intensity of the images acquired after initial staining and restaining were compared. A number of 3-D (texture) features computed after segmentation of nuclei were compared as well. The best destaining result was obtained by incubation of sections at 37 degrees C in preheated medium twice for 20 min. On average, the 3-D feature values were comparable with those after initial staining. With the described protocol it is possible to remove TO-PRO-3 fluorescence from tissue sections that can successfully be restained with minimal influence on fluorescence intensity and nuclear chromatin distribution. |
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Authors:
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André Huisman; Lennert S Ploeger; Hub F J Dullens; Jan T C Beekhuis; Paul J van Diest |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Cytometry. Part A : the journal of the International Society for Analytical Cytology Volume: 71 ISSN: 1552-4922 ISO Abbreviation: Cytometry A Publication Date: 2007 Oct |
Date Detail:
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Created Date: 2007-09-26 Completed Date: 2007-12-07 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101235694 Medline TA: Cytometry A Country: United States |
Other Details:
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Languages: eng Pagination: 875-81 Citation Subset: IM |
Affiliation:
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Department of Pathology, University Medical Center, Utrecht, The Netherlands. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Carbocyanines
/
analysis,
chemistry Chromatin / metabolism Fluorescence Microscopy, Confocal / methods* Staining and Labeling / methods* |
| Chemical | |
Reg. No./Substance:
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0/Carbocyanines; 0/Chromatin; 0/TO-PRO-3 |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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