ARTICLE |
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
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, ammoniaethanol, 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. Ammoniaethanol 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. Ammoniaethanol 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 49:15651571, 2001)
Key Words: immunofluorescence, autofluorescence, background fluorescence, confocal laser scanning, microscopy, paraffin sections, archival tissue, formaldehyde, bone marrow
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conventional fluorescence microscopy is a powerful tool for both morphological studies and clinical diagnosis. However, photographic documentation of fluorescence microscopic findings is restricted to thin sections in which interference with out-of-focus fluorescence is irrelevant. Microscopic analysis of three-dimensional tissue patterns requires thicker sections, and for visual evaluation the plane of focus has to be moved by hand across the full thickness of the section. In contrast, confocal laser scanning fluorescence microscopy (CLSM), in which interference with out-of-focus light is shielded off, has no such restrictions. It can be operated at quasi-theoretical resolution provided that unwanted fluorescence is absent. Unwanted fluorescence is either due to autofluorescence of the tissue of interest or presents as a fluorescent background caused by nonspecific binding of the fluorescent label.
Autofluorescent properties of specific tissue constituents may be of diagnostic value (
Various histochemical techniques for the removal of autofluorescence have evolved. Ammoniaethanol has been applied to remove some of the formaldehyde-derived artifacts (
Here we assessed the effects of the three reagents, ammoniaethanol, borohydride, and sudan on autofluorescence in paraffin sections of archival formaldehyde-fixed and decalcified human bone marrow before fluorescence labeling. We compared them with the effects on archival myocardium and cartilage and with frozen sections of the same tissue types. The aim of our study was to minimize autofluorescence to enable high-resolution CLSM also in formaldehyde-fixed archival tissue and to elucidate the influence of tissue type, fixation medium, and tissue processing.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unless otherwise specified, all reagents were of analytical grade and purchased from Sigma (St Louis MO), Fluka (Buchs, Switzerland), or Merck (Darmstadt, Germany), and all steps of the procedures were done at ambient temperature (2025C).
Tissue Sections
Paraffin blocks, stored for 6 months to 3 years, were retrieved from our archival files of human bone marrow and myocardial biopsies fixed in neutral buffered formaldehyde (4% final concentration) for 1224 hr, cut to 8-µm-thick sections, and mounted on sialinized slides. After fixation, bone marrow core biopsies were decalcified overnight in 5% trichloroacetic acid under continuous agitation. Cartilage specimens, a gift from J. Seidel (MIT; Cambridge MA), were derived from articular bovine chondrocytes grown on a scaffold of polyglycolic acid (
Frozen sections of postmortem human bone marrow taken from the femoral medulla required no decalcification. They were cut to 8-µm thickness, postfixed for 1 hr either in formaldehyde (4% final concentration) or in 2.5% glutaraldehyde, and air-dried until further processing.
Sections were treated with ammoniaethanol, borohydride, or sudan and by combinations thereof as follows.
AmmoniaEthanol
While rehydrating the deparaffinized sections in graded alcohol, the slides were immersed for 1 hr in 70% ethanol supplemented with 0.25% NH3, and rehydration was resumed by immersion in 50% ethanol for 10 min, after which the sections were transferred to MHB.
Borohydride
Deparaffinized sections were immersed in ice-cooled freshly prepared MHB supplemented with 10 mg/ml borohydride for 40 min, washed three or four times in MHB, and stored at 4C in MHB until further processing.
Frozen sections were treated accordingly after permeabilization as described above.
Sudan
After fluorescence labeling, the slides were immersed for 30 min in 70% ethanol supplemented with 0.1% Sudan Black B (Merck). The removal of excess sudan from the slides proved decisive. Sudan had to be wiped off manually from the back and along the edges of the slide with a soft paper, and the front required a jet wash with MHB. Subsequently, slides were immersed for 10 min in MHB and mounted. Omitting this procedure resulted in sudan precipitates appearing as subcellular black grains in the differential interference contrast (DIC) image.
Postfixed frozen sections were treated accordingly for autofluorescence control, observing the sequential order of borohydride, ammoniaethanol, and sudan.
Immunofluorescence Labeling
The slides stored in MHB were conditioned for 20 min with 5075 µl of MHB containing 10% swine serum (Jackson Immune Research Laboratories; West Grove, PA) and incubated for 1 hr with the primary antibody diluted in MHB. The sections were washed three or four times for 5 min with aliquots of 100200 µl of MHB, incubated for 1 hr with secondary antibody diluted in 75 µl MHB, and washed as above. Primary antibodies used were directed against ß-tubulin (clone KMX-1, 1:50; Boehringer, Mannheim, Germany) and the secondary antibody was donkey anti-mouse labeled by Cy2 (1:400, 488 nm; Amersham, Poole, UK).
All sections were mounted with Mowiol-1188 (Hoechst; Frankfurt, Germany) supplemented with 0.75% of the anti-fading agent n-propyl-gallate (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone Marrow Paraffin Sections
In untreated paraffin sections of formaldehyde-fixed, decalcified bone marrow, trabecular bone and cytoplasmic granules of myeloid cells displayed autofluorescence (Fig 1A and Fig 1B). Treatment with ammoniaethanol reduced bright autofluorescence of myeloid cells most efficiently regardless of the wavelength (488 nm, 568 nm, or 647 nm) used for excitation (Fig 1C). Reduction of autofluorescence was observed at 488 nm and 568 nm also after treatment with sudan (Fig 1D), but not at 647 nm, where sudan's own fluorescence became apparent (
|
The complex organotypical histoarchitecture of the bone marrow was more accurately appreciated in DIC, where trabecular bone, cells of various hematopoietic cell lines, and stromal structures, including adipocytes, were easily distinguishable (Fig 2A).
|
Bone Marrow Frozen Sections
In untreated formaldehyde-postfixed frozen sections of non-decalcified bone marrow, autofluorescent cytoplasmic granules were seen in myeloid cells (Fig 2C), similar to decalcified paraffin sections (Fig 2B). Sudan decreased unwanted autofluorescence most effectively (not shown), again when exposed at 488-nm and 568-nm wavelengths, but not at 647 nm, for which ammoniaethanol proved superior. Borohydride was inefficient in this respect (Fig 2C and Fig 2F).
In glutaraldehyde-postfixed untreated frozen sections of non-decalcified bone marrow, myeloid cells displayed bright autofluorescence (Fig 2D). The latter was considerably reduced by borohydride (Fig 2E), and occasionally even more so when combined with ammoniaethanol and/or sudan (not shown).
Myocardial Paraffin Sections
In untreated paraffin sections of formaldehyde-fixed myocardium, lipofuscin granules showed as autofluorescent particles, whereas the sarcoplasmic body of myocardiocytes remained dim (Fig 2G). Treatment with ammoniaethanol and borohydride, either separately or combined, highlighted the sarcomeric Z-lines at 488 nm and 568 nm (Fig 2H). They were not perceivable at 647 nm (not shown), whereas autofluorescence of lipofuscin granules was present at all three wavelengths. Treatment with sudan, however, completely removed autofluorescence from myocardiocytes (Fig 2I).
Cartilage Paraffin Sections
In untreated paraffin sections of engineered bovine cartilage, weak autofluorescence was present in the intercellular space. It was related to the scaffold made of polyglycolic acid, which was used for cultivation. Whereas borohydride and ammoniaethanol induced cytoplasmic fluorescence in chondrocytes, optimal control of autofluorescence was achieved by combining no less than all three reagents, i.e., ammoniaethanol, borohydride, and sudan (not shown).
Interference with Fluorescence Labeling
To illustrate the applicability of autofluorescence control, an example of a myeloproliferative disorder was selected from our archival bone marrow files and labeled for ß-tubulin immunofluorescence. In tubulin-labeled untreated paraffin sections exposed at 488 nm, an ill-defined cluster of green fluorescence was registered (Fig 3A, arrow). When the same area was exposed at 568 nm, distinct autofluorescence was displayed by cytoplasmic granules in myeloid cells (Fig 3B, arrow). The presence of these autofluorescent granules at 488 nm was camouflaged by the immunofluorescent label (Fig 3A, arrow). After treatment with ammoniaethanol and sudan, stromal cells and myeloid cells displayed a green fluorescent cytoplasm, indicating the presence of tubulin (Fig 3C), whereas unwanted autofluorescence was absent (Fig 3D). In conjunction with proper autofluorescence control, CLSM provided a high-resolution insight into the cytoskeletal architecture of microtubules in both interphase (Fig 3C') and mitotic cells (Fig 3C'').
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interference by autofluorescence is one of the major shortcomings of CLSM in immunofluorescence analysis. This phenomenon may either be intrinsic, due to fluorescent structures in cells and interstitial tissue, or may be induced by fixation media and tissue processing techniques. Yet another source of unwanted fluorescence relates to nonspecific binding of the fluorescent marker (
At least three different strategies can be distinguished in the control of autofluorescence: (a) extraction of the autofluorescent constituent by dissolution; (b) chemical modification of the fluorochrome; and (c) masking of the autofluorescent structure by appropriate staining.
Ammoniaethanol may promote the removal of precipitates formed during bone decalcification by trichloroacetic acid. It may dissolve negatively charged lipid derivatives, phenols or polypenols, and degrade weak esters by hydrolysis. Improved extraction and dissolution by ammoniaethanol of autofluorescent entities are the putative modes of action.
Borohydride interacts with aldehydes and ketones by reducing both reactive groups to the respective alcohols. Apart from its usefulness in removing the excess of aldehyde after tissue fixation, borohydride also efficiently reduces processing-related fluorescence in glutaraldehyde-fixed tissue (
Unlike ammoniaethanol and borohydride, sudan masks autofluorescent structures (
With respect to the difference of action among the three agents tested, there is no surprise at their variable effects depending on tissue type, fixation, processing, and wavelength of excitation light. The comparative assessment of ammoniaethanol, borohydride, and sudan on sections cut from the same tissue blocks should allow a ranking of the agents' performances.
Ammoniaethanol proved most efficient on paraffin sections of formaldehyde-fixed, decalcified bone marrow, but much less so on frozen sections of non-decalcified bone marrow. Its effect on autofluorescent granules of myeloid cells was obvious, but its performance against fluorescence in formaldehyde-fixed bone marrow and myocardium was far below our expectations. Introduced by Kardasewitsch in 1925, ammoniaethanol proved nevertheless a powerful suppressor of autofluorescence in paraffin sections of formaldehyde-fixed, decalcified bone marrow. Deposits formed during decalcification by trichloroacetic acid and formaldehyde-induced condensates may be thoroughly dissolved (
Borohydride is now a confirmed requisite for autofluorescence control in glutaraldehyde-fixed tissue (
Sudan was generally effective for fluorescence control in formaldehyde-postfixed frozen sections of bone marrow and in myocardial paraffin sections. Masking of brightly fluorescent lipofuscin granules by sudan is observed at a concentration as low as 0.1% in our experience, but may require a higher concentration of 1% in neural tissue (
The results of the present study highlight the fact that there is no general recipe available for the control of autofluorescence. Success was found in a tactical approach that can be summarized as choice of the appropriate reagent(s) by trial and error, either single or combined, mutually designed for the specific tissue type, fixation medium, processing technique, and wavelength of excitation light.
Thus far, CLSM has been applied on routine paraffin sections for visualizing the three-dimensional architecture by staining cells with fluorescent dyes (
![]() |
Acknowledgments |
---|
Supported by the Swiss Cancer League grant no. SKL-00653-2-1998/ KFS 962-9-1999.
We thank Dr L. Landmann (Institute of Anatomy, University of Basel) for the complimentary use of CLSM facilities.
Received for publication February 17, 2001; accepted June 27, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Banerjee B, Miedema BE, Chandrasekhar HR (1999) Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon. J Invest Med 47:326-331[Medline]
Baschong W, Sütterlin R, Aebi U (1997) Punch-wounded, fibroblast-populated collagen matrices. A novel approach for studying cytoskeletal changes in 3-dimensions by confocal laser scanning microscopy. Eur J Cell Biol 72:189-201[Medline]
Baschong W, Suetterlin R, Hefti A, Schiel H (2001) Confocal laser scanning microscopy and scanning electron microscopy of tissue ti-implant interfaces. Micron 32:33-41[Medline]
Baschong W, Dürrenberger M, Mandinova A, Suetterlin R (1999) Three dimensional visualization of the cytoskeleton by confocal laser scanning microscopy. In Cohn PM, ed. Confocal Microscopy. Methods in Enzymology. Vol 307. San Diego, Academic Press, 173-189
Belichenko PV, Fedorov AA, Dahlström AB (1996) Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal scanning microscopy. J Neurosci Methods 69:155-161[Medline]
Brotchie D, Birch M, Roberts N, Howard CV, Smith VA, Grierson I (1999) Localization of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy. J Neurosci Methods 87:77-85[Medline]
Del Castillo P, Llorente AR, Stockert JC (1989) Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis. Bas Appl Histochem 33:251-257
Edwin EE, Jackman R (1981) Nature of autofluorescent material in cerebrocortical necrosis. J Neurochem 37:1054-1056[Medline]
Freed LE, Hollander AP, Martin I, Barry JR, Langer R, VunjakNovakovic G (1998) Chondrogenesis in a cell-polymer-bioreactor system. Exp Cell Res 240:58-65[Medline]
Johnson TAA (1987) Glutaraldehyde fixation chemistry: oxygen-consuming reactions. Eur J Cell Biol 45:160-169[Medline]
Kardasewitsch B (1925) Eine Methode zur Beseitigung der Formalin-sedimente (Paraform) aus mikroskopischen Praeparaten. Zeitsch Wissenschr Mikrosk Techn 42:322-324
Kittelberger R, David PF, Stehbens WI (1989) An improved immunofluorescence technique for the histological examination of blood vessel tissue. Acta Histochem 86:137-142[Medline]
Liu S, Weaver DL, Taatjes DJ (1997) Three-dimensional reconstruction by confocal laser scanning microscopy in routine pathologic specimens of benign and malignant lesions of the human breast. Histochem Cell Biol 107:267-278[Medline]
Noonberg SB, Weiss TL, Garovoy MR, Hunt CA (1992) Characterization and minimization of cellular autofluorescence in the study of oligonucleotide uptake using confocal microscopy. Antisense Res Dev 2:303-333[Medline]
Romijn HJ, van Uum JFM, Bredijk I, Emmering J, Radu I, Pool CW (1999) Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy. J Histochem Cytochem 47:229-235
Schnell StA, Staines WA, Wessendorf MW (1999) Reduction of liposfuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47:719-730
Small JV, Celis JE (1978) Direct visualization of the 10 nm (100 Å)-filament network in whole and enucleated cultured cells. J Cell Sci 31:393-409[Abstract]
Southern LJ, Hughes H, Lawford PV, Clench MR, Maning NJ (2000) Glutaraldehyde-induced crosslinks: a study of model compounds and commercial bioprosthetic valves. J Heart Valve Dis 9:241-248[Medline]
Tekola P, Baak JPA, Bellien JAM, Brugghe J (1994) Highly sensitive, specific and stable new fluorescent DNA stains for confocal laser scanning microscopy and image processing of normal paraffin sections. Cytometry 17:191-195[Medline]
Verbunt RJAM, Fitzmaurice MA, Kramer JR, Ratcliff NB, Kittrell C, Taroni P, Cothren RM, Baraga J, Feld M (1992) Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery. Am Heart J 123:208-216[Medline]