ARTICLE

Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

Correspondence to: Werner Baschong, M. E. Muller Institute at the Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel CH-4056, Switzerland. E-mail: Werner.Baschong@unibas.ch

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia–ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia–ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia–ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components. (J Histochem Cytochem 49:1565–1571, 2001)

Key Words: immunofluorescence, autofluorescence, background fluorescence, confocal laser scanning, microscopy, paraffin sections, archival tissue, formaldehyde, bone marrow

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. Pilling, D. Roife, M. Wang, S. D. Ronkainen, J. R. Crawford, E. L. Travis, and R. H. Gomer Reduction of Bleomycin-Induced Pulmonary Fibrosis by Serum Amyloid P J. Immunol., September 15, 2007; 179(6): 4035 - 4044. [Abstract] [Full Text] [PDF]

				R. J. Byers, D. Di Vizio, F. O'Connell, E. Tholouli, R. M. Levenson, K. Gossard, D. Twomey, Y. Yang, E. Benedettini, J. Rose, et al. Semiautomated Multiplexed Quantum Dot-Based in Situ Hybridization and Spectral Deconvolution J. Mol. Diagn., February 1, 2007; 9(1): 20 - 29. [Abstract] [Full Text] [PDF]

				J. Tian, R. Mahmood, R. Hnasko, and J. Locker Loss of Nkx2.8 Deregulates Progenitor Cells in the Large Airways and Leads to Dysplasia Cancer Res., November 1, 2006; 66(21): 10399 - 10407. [Abstract] [Full Text] [PDF]

				C. N. Craciunescu, R. Wu, and S. H. Zeisel Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus FASEB J, August 1, 2006; 20(10): 1635 - 1640. [Abstract] [Full Text] [PDF]

				P. Kanellakis, N. J. Slater, X.-J. Du, A. Bobik, and D. J. Curtis Granulocyte colony-stimulating factor and stem cell factor improve endogenous repair after myocardial infarction Cardiovasc Res, April 1, 2006; 70(1): 117 - 125. [Abstract] [Full Text] [PDF]

				D. E. Ferrara, D. Weiss, P. H. Carnell, R. P. Vito, D. Vega, X. Gao, S. Nie, and W. R. Taylor Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R114 - R123. [Abstract] [Full Text] [PDF]

				L. B. Ngwenya, A. Peters, and D. L. Rosene Light and Electron Microscopic Immunohistochemical Detection of Bromodeoxyuridine-labeled Cells in the Brain: Different Fixation and Processing Protocols J. Histochem. Cytochem., July 1, 2005; 53(7): 821 - 832. [Abstract] [Full Text] [PDF]

				R. Suetterlin, W. Baschong, and R. H. Laeng Immunofluorescence and Confocal Laser Scanning Microscopy of Chronic Myeloproliferative Disorders on Archival Formaldehyde-fixed Bone Marrow J. Histochem. Cytochem., March 1, 2004; 52(3): 347 - 354. [Abstract] [Full Text] [PDF]

				C. N. Craciunescu, C. D. Albright, M.-H. Mar, J. Song, and S. H. Zeisel Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus J. Nutr., November 1, 2003; 133(11): 3614 - 3618. [Abstract] [Full Text] [PDF]

				J. M. Levsky and R. H. Singer Fluorescence in situ hybridization: past, present and future J. Cell Sci., July 15, 2003; 116(14): 2833 - 2838. [Abstract] [Full Text] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2001