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Triple immunological staining with colloidal gold, fluorescein and rhodamine as labels
Journal oflmmunologicalMethods, 80 (1985) 1-6
1
Elsevier JIM03394
Triple Immunological Staining with Colloidal Gold, Fluorescein and Rhodamine as Labels i J.J.M. Van Dongen 2, H. Hooijkaas, W.M. Comans-Bitter, K. Benne, T.M. Van Os and J. De Josselin de Jong Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands
(Received 7 August 1984, accepted 1 November 1984)
Colloidal gold particles are detectable by light microscopy with polarized light in the same epi-illumination system as for fluorescence microscopy. Colloidal gold particles can thus be used in combination with a fluorochrome for the combined immunological detection of surface membrane and cytoplasmic markers. We analyzed human bone marrow cells by a triple immunological staining for 3 different markers, using colloidal gold, fluorescein and rhodamine as labels. Our results demonstrate that such a triple immunological staining provides a powerful tool for study of the heterogeneity of small cell populations. Key words: triple immunological staining - colloidal gold - epi -illumination - fluorescence microscopy
Introduction In m i c r o s c o p y , i m m u n o l o g i c a l staining techniques are widely used for b o t h research a n d d i a g n o s t i c purposes, especially for the d e t e c t i o n of surface m e m b r a n e a n d c y t o p l a s m i c markers. T h e a n t i b o d i e s used can b e c o n j u g a t e d with fluorochrom e s such as fluorescein i s o t h i o c y a n a t e ( F I T C ) o r t e t r a m e t h y l r h o d a r n i n e isot h i o c y a n a t e ( T R I T C ) , o r with enzymes such as p e r o x i d a s e (PO) o r alkaline p h o s p h a t a s e (AP). F o r the s i m u l t a n e o u s d e t e c t i o n o f different markers, a d o u b l e staining technique c a n be used, e.g,, F I T C in c o m b i n a t i o n with T R I T C (Ploem, 1967) o r P O in c o m b i n a t i o n with A P (Feller a n d Parwaresch, 1983; M o i r et al., 1983).
i This work was supported by the Netherlands Cancer Foundation (Koningin Wilhelmina Fonds). z Correspondence to: Dr. J.J.M. Van Dongen, Dept. Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Abbreviations: AP, alkaline phosphatase; BM¢ bone marrow; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; McAb, monoclonal antibody; MNC, mononuclear cells; PBS, phosphate-buffered saline; PO, peroxidase; RT, room temperature; SITS, acetamide isothiocyanate stilbene disulphonic acid; T-ALL T cell acute lymphoblastic leukemia; TdT, terminal deoxynucleotidyl transferase; TRITC, tetramethylrhodamine isothiocyanate. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Double staining techniques provide additional information about marker distribution within a cell population that cannot be obtained by 2 separate stainings of individual samples. For instance, the combined detection of kappa and lambda immunoglobulin (Ig) light chains on different cells proves the B cell character of these cells, while their presence on the same cell indicates Fc receptor binding of autologous Ig, e.g., by monocytes (Schuit and Hijmans, 1980). Another example of the useful application of double staining is the simultaneous detection of a T cell marker and the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) on single cells. Since T cell marker+/TdT + cells normally occur only in the thymus the presence of cells with the T cell marker+/TdT + phenotype in extra-thymic locations is indicative of T cell acute lymphoblastic leukemia (T-ALL) or TdT-positive T cell non-Hodgkin lymphoma (Bradstock et al., 1981; Van Dongen et al., 1984). For the identification of small cell populations, it may be mandatory to use a triple staining technique for detection of 3 different markers at the single cell level. In microscopy it is difficult to combine FITC and TRITC with a third fluorochrome, e.g., SITS (Rothbarth et al., 1978), Texas Red (Titus et al., 1982) or phycoerythrin (Oi et al., 1982), because of marked fading or a considerable overlap in excitation and emission spectra (Shapiro, 1983). Recently a technique was described which allows the detection of colloidal gold particles in light microscopy by epi-illumination with polarized light (De Mey, 1983). In theory, colloidal gold particles are a suitable label for combining with fluorochrorues. We adapted this technique for triple staining with colloidal gold, F1TC and TRITC.
Materials and Methods
Preparation of cell suspension Bone marrow (BM) cells from children treated for non-T-ALL and from healthy adul.t volunteers were obtained by needle aspiration. Mononuclear cells (MNC) were isolated by Ficoll-Paque (density 1.077 g/cm3; Pharmacia, Uppsala) density centrifugation (B6yum, 1968). The MNC were washed twice. These and all subsequent washings of cell suspensions were performed with phosphate-buffered saline (PBS) (300 mosmol; pH 7.8), supplemented with 1% bovine serum albumin (BSA; Organon Teknika, Oss) and 0.05% sodium azide. The cell concentration was adjusted to 107 cells per ml.
Triple staining technique We performed triple staining for the detection of the T cell marker 3A1 (Haynes et al., 1980), a non-polymorphic HLA-DR antigen (Lampson and Levy, 1980) and the nuclear enzyme TdT. Fifty microlitres of the cell suspension were incubated with 50 pl of the optimally titrated monoclonal antibody (McAb) 3A1 (American Type Culture Collection, Rockville MD) at room temperature (RT). After 30 rain the cells were washed twice and incubated (30 rain, RT) with 50 pl undiluted goat anti-mouse Ig antiserum, conjugated with colloidal gold particles of 30 nm (G30; Janssen
Pharmaceutica, Beerse). The cells were washed twice and subsequently incubated (30 rain, RT) with normal mouse serum (diluted 1 in 100 in PBS) to block free antigen binding rites of the goat anti-mouse Ig serum. The cells were washed twice and incubated (30 rain, RT) with a TRITC-conjugated anti-HLA-DR McAb (Becton Dickinson, Sunnyvale, CA). After 2 washings the cell suspension was adjusted to 2 x 106 cells per ml. Fifty microlitres of this ~ell suspension were centrifuged on slides in a cytocentrifuge (Nordic, Tilburg). The cytocentrifuge preparations were air dried during 15 min and the location of the cells was marked by encircling with a glass pencil. The preparations were fixed in methanol (30 min, 4°C) and washed in PBS for 15 min. The slides were dried and incubated with 15/tl of a rabbit anti-TdT antiserum (Bethesda Research Laboratory, Gaithersburg, MD) in a moist chamber (30 min, RT). The slides were then washed in PBS for 15 min, dried and incubated with 15 /~1 FITC-conjugated goat anti-rabbit Ig antiserum (Bethesda Research Laboratory) in a moist chamber (30 rain, RT). The slides were washed again in PBS (15 rain), dried and mounted in glycerol/PBS (9:1) containing 1 mg phenylenediamine/ml to prevent fading of the fluorochromes (Johnson and Nogueira Araujo, 1981). A coverslip was sealed to the slide with paraffin wax with ceresin (BDH Chemicals, Poole, England).
Microscopes For evaluation of immunofluorescence and immunogold staining Zeiss and Leitz fluorescence microscopes were used. Both were equipped with phase contrast facilities and an adjustable aperture diaphragm in the epi-illumination system. The Zeiss microscope (Carl Zeiss, Oberkochen) was a Zeiss Universal !I, equipped with a III RS Fluoreszenz-Auflicht condenser and a 50 W HBO mercury lamp (Osram, Berlin) and two 63 × objectives (Carl Zeiss): neofluar 63/1.25 Oel Ph 3 and neofluar 63/1.25 Oel. Filter combination 19 (Carl Zeiss) was used for the evaluation of FITC and filter combination 14 (Carl Zeiss) for TRITC. The Leitz microscope (Ernst Leitz Wetzlar, Wetzlar) was a Leitz Orthoplan, equipped with a Leitz Ploemopak 2 illumination, a 100 W HBO mercury lamp (Osram), a Leitz Vario Orthomat camera and two 63 × objectives (Leitz): PL APO 63/1.40 Oel Phaco 4 and 63/1.30 Oel Fluoreszenz. Filter combination I2 (Leitz) was used for evaluation of FITC and filter combination N2 (Leitz) for TRITC. For evaluation of colloidal gold, we used in both microscopes a polarization filter combination, consisting of a polarizer for the excitation light, a beamsplitter and an analyzer that extinguished reflected light by having the same polarization as the excitation light (De Mey, 1983).
Results
Microscopes 63 x objectives with phase rings can be used for visualization of FITC and TRITC, but not for colloidal gold because of high background, probably due tO reflection and depolarization of the polarized light by the phase rings. Visualization of the colloidal gold was made possible by the use of 63 × objectives without phase
rings and by closing the aperture diaphragm of the epi-illumination system. With the phase contrast dia-illumination system, the gold particles were not detectable, but by epi-illumination with polarized light the gold-yellow scattered light was clearly visible. For routine evaluation of triple staining the Zeiss microscope was used.
Triple immunological staining In the BM samples tested the percentage TdT positive cells ranged from 0.5% to 10%. The triple staining revealed that the majority (more than 70%) of the TdT positive cells was positive for HLA-DR, but negative for the T cell marker 3A1 (Fig. 1). A minority (0.1-8%) of the TdT positive cells was positive for both H L A - D R and 3A1. Detailed studies of these cell populations will be published elsewhere.
Discussion
Colloidal gold particles are microscopically detectable by polarized light used in the same epi-illumination system as for fluorescence. Our results demonstrate that in triple immunological staining, colloidal gold particles can be used as a third label in combination with the fluorochromes FITC and TRITC. The triple staining of BM cells for 3A1, H L A - D R and TdT revealed that within the small TdT positive cell population subpopulations are present, e.g. a 3 A 1 - / H L A - D R + / T d T + subpopulation, and also a very small one of 3A1 + / H L A - D R + / T d T ÷ phenotype. Thus triple immunological staining can be a powerful tool in analyzing the heterogeneity of small cell populations in cell differentiation studies. Since we used 2 mouse McAb and a rabbit antiserum in the triple staining, it was sufficient to use only 1 conjugated McAb. However, where combination of 3 mouse McAb is required, it has hitherto been necessary to use at least 2 conjugated McAb or McAb differing in Ig (sub)class in combination with different conjugated antimouse Ig (sub)class antisera. A more elegant and less time-consuming solution is to use 3 differently conjugated McAb. Therefore it would be convenient if the McAb of the required specificities were not only available as FITC or T R I T C conjugates, but also as colloidal gold conjugates.
Acknowledgements
We gratefully acknowledge Prof. Dr. R. Benner's support, and thank Mrs. E.G.M. Cristen, A.A. van der Linde-Preesman and I.L.M. Tettero for their excellent technical assistance, Dr. J. De Mey from Janssen Pharmaceutica and Dr. J.H.W. Leuvering Fig. 1. Triple immunologicalstaining of BM cells for 3AI, HLA-DR and TdT. a: phase contrast; b: 3A1 positive cells (colloidalgold labelled); c: HLA-DR positivecells (TRITC labelled); d: TdT positive cells (FITC labelled). The 2 TdT positive cells are also positive for HLA-DR, but not for the T cell marker 3A1; they probably representprecursor B cells. Three cells are positivefor 3A1 and not for HLA-DR and TdT, probably representingnormal T lymphocytes.
f r o m O r g a n o n I n t e r n a t i o n a l B.V. a n d Dr. M. D e W a e l e for their advice, Mr. C . G . E n k e l a a r f r o m Z e i s s N e d e r l a n d for his t e c h n i c a l s u p p o r t , Mr. J. F e n g l e r for his p h o t o g r a p h i c assistance, a n d Mrs. C . J . M . M e i j e r i n k - C l e r k x for t y p i n g this manuscript.
N o t e Added in P r o o f T h e e v a l u a t i o n o f the c o l l o i d a l g o l d s t a i n i n g c a n be i m p r o v e d b y use of the Z e i s s objective Antiflex-Neofluar 63/1.25 Oel which contains a rotable quarter-wave plate.
References B0yum, A., 1968, Scand. J. Clin. Lab. Invest. 21, 77. Bradstock, K.F., G. Janossy, N. Tidman, E.S. Papageorgiou, H.G. Prentice, M. Willoughby and A.V. Hoffbrand, 1981, Leuk. Res. 5, 301. De Mey, J., 1983, in: Immunocytochemistry, Practical Application in Pathology and Biology, eds. J,M. Polak and S. Van Noorden (Wright PSG, Bristol) p. 82. Feller, A.C. and M.R. Parwaresch, 1983, J. Immunol. Methods 63, 273. Haynes, B.F., D.L. Mann, M.E. Hemler, J.A. Schroer, J.H. Shelhamer, G.S. Eisenbarth, J.L. Strominger, C.A. Thomas, H.S. Mostowski and A.S. Fa~uci, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 2914. Hijmans, W., J.J. Haaijman and H.R.E. Schuit, 1981, in: Immunological Techniques Applied to Aging Research, eds. W.H. Adler and A.A. Nordin (CRC Press, Boca Rato, FL) p. 141. Johnson, G.D. and G.M. de C. Nogueira Araujo, 1981, J. Immunol. Methods 43, 349. Lampson, L.A. and R. Levy, 1980, J. Immunol. 125, 293. Moir, D.J., A.K. Ghosh, Z. Abdulaziz, P.M. Knight and D.Y. Mason, 1983, Br. J. Haematol. 55, 395. Oi, V.T., A.N. Glazer and L. Stryer, 1982, J. Cell Biol. 93, 981. Ploem, J.S., 1967, Z. Wiss. Mikr. 68, 129. Rothbarth, Ph.H., H.J. Tanke, N.A.J. Mul, J.S. Ploem, J.F.G. Vliegenthart and R.E. Ballieux, 1978, J. Immunol. Methods 19, 101. Schuit, H.R.E. and W. Hijmans, 1980, Clin. Exp. Immunol. 41,567. Shapiro, H.M., 1983, Cytometry 3, 227. Titus, J.A., R. Haugland, S.O. Sharrow and D.M. Segal, 1982, J. Immunol. Methods 50, 193. Van Dongen, J.J.M., H. Hooijkaas, K. H~.hlen, K. Benne, W.M. Bitter, A.A. Van der Linde-Preesman, I.L.M. Tettero, M. Van de Rijn, J. Hilgers, G.E. Van Zanen and A. Hagemeijer, 1984, in: Minimal Residual Disease in Acute Leukemia, eds. B. L6wenberg and A. Hagenbeek (Martinus Nijhoff, Boston) p. 67.
1
Elsevier JIM03394
Triple Immunological Staining with Colloidal Gold, Fluorescein and Rhodamine as Labels i J.J.M. Van Dongen 2, H. Hooijkaas, W.M. Comans-Bitter, K. Benne, T.M. Van Os and J. De Josselin de Jong Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands
(Received 7 August 1984, accepted 1 November 1984)
Colloidal gold particles are detectable by light microscopy with polarized light in the same epi-illumination system as for fluorescence microscopy. Colloidal gold particles can thus be used in combination with a fluorochrome for the combined immunological detection of surface membrane and cytoplasmic markers. We analyzed human bone marrow cells by a triple immunological staining for 3 different markers, using colloidal gold, fluorescein and rhodamine as labels. Our results demonstrate that such a triple immunological staining provides a powerful tool for study of the heterogeneity of small cell populations. Key words: triple immunological staining - colloidal gold - epi -illumination - fluorescence microscopy
Introduction In m i c r o s c o p y , i m m u n o l o g i c a l staining techniques are widely used for b o t h research a n d d i a g n o s t i c purposes, especially for the d e t e c t i o n of surface m e m b r a n e a n d c y t o p l a s m i c markers. T h e a n t i b o d i e s used can b e c o n j u g a t e d with fluorochrom e s such as fluorescein i s o t h i o c y a n a t e ( F I T C ) o r t e t r a m e t h y l r h o d a r n i n e isot h i o c y a n a t e ( T R I T C ) , o r with enzymes such as p e r o x i d a s e (PO) o r alkaline p h o s p h a t a s e (AP). F o r the s i m u l t a n e o u s d e t e c t i o n o f different markers, a d o u b l e staining technique c a n be used, e.g,, F I T C in c o m b i n a t i o n with T R I T C (Ploem, 1967) o r P O in c o m b i n a t i o n with A P (Feller a n d Parwaresch, 1983; M o i r et al., 1983).
i This work was supported by the Netherlands Cancer Foundation (Koningin Wilhelmina Fonds). z Correspondence to: Dr. J.J.M. Van Dongen, Dept. Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Abbreviations: AP, alkaline phosphatase; BM¢ bone marrow; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; McAb, monoclonal antibody; MNC, mononuclear cells; PBS, phosphate-buffered saline; PO, peroxidase; RT, room temperature; SITS, acetamide isothiocyanate stilbene disulphonic acid; T-ALL T cell acute lymphoblastic leukemia; TdT, terminal deoxynucleotidyl transferase; TRITC, tetramethylrhodamine isothiocyanate. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Double staining techniques provide additional information about marker distribution within a cell population that cannot be obtained by 2 separate stainings of individual samples. For instance, the combined detection of kappa and lambda immunoglobulin (Ig) light chains on different cells proves the B cell character of these cells, while their presence on the same cell indicates Fc receptor binding of autologous Ig, e.g., by monocytes (Schuit and Hijmans, 1980). Another example of the useful application of double staining is the simultaneous detection of a T cell marker and the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) on single cells. Since T cell marker+/TdT + cells normally occur only in the thymus the presence of cells with the T cell marker+/TdT + phenotype in extra-thymic locations is indicative of T cell acute lymphoblastic leukemia (T-ALL) or TdT-positive T cell non-Hodgkin lymphoma (Bradstock et al., 1981; Van Dongen et al., 1984). For the identification of small cell populations, it may be mandatory to use a triple staining technique for detection of 3 different markers at the single cell level. In microscopy it is difficult to combine FITC and TRITC with a third fluorochrome, e.g., SITS (Rothbarth et al., 1978), Texas Red (Titus et al., 1982) or phycoerythrin (Oi et al., 1982), because of marked fading or a considerable overlap in excitation and emission spectra (Shapiro, 1983). Recently a technique was described which allows the detection of colloidal gold particles in light microscopy by epi-illumination with polarized light (De Mey, 1983). In theory, colloidal gold particles are a suitable label for combining with fluorochrorues. We adapted this technique for triple staining with colloidal gold, F1TC and TRITC.
Materials and Methods
Preparation of cell suspension Bone marrow (BM) cells from children treated for non-T-ALL and from healthy adul.t volunteers were obtained by needle aspiration. Mononuclear cells (MNC) were isolated by Ficoll-Paque (density 1.077 g/cm3; Pharmacia, Uppsala) density centrifugation (B6yum, 1968). The MNC were washed twice. These and all subsequent washings of cell suspensions were performed with phosphate-buffered saline (PBS) (300 mosmol; pH 7.8), supplemented with 1% bovine serum albumin (BSA; Organon Teknika, Oss) and 0.05% sodium azide. The cell concentration was adjusted to 107 cells per ml.
Triple staining technique We performed triple staining for the detection of the T cell marker 3A1 (Haynes et al., 1980), a non-polymorphic HLA-DR antigen (Lampson and Levy, 1980) and the nuclear enzyme TdT. Fifty microlitres of the cell suspension were incubated with 50 pl of the optimally titrated monoclonal antibody (McAb) 3A1 (American Type Culture Collection, Rockville MD) at room temperature (RT). After 30 rain the cells were washed twice and incubated (30 rain, RT) with 50 pl undiluted goat anti-mouse Ig antiserum, conjugated with colloidal gold particles of 30 nm (G30; Janssen
Pharmaceutica, Beerse). The cells were washed twice and subsequently incubated (30 rain, RT) with normal mouse serum (diluted 1 in 100 in PBS) to block free antigen binding rites of the goat anti-mouse Ig serum. The cells were washed twice and incubated (30 rain, RT) with a TRITC-conjugated anti-HLA-DR McAb (Becton Dickinson, Sunnyvale, CA). After 2 washings the cell suspension was adjusted to 2 x 106 cells per ml. Fifty microlitres of this ~ell suspension were centrifuged on slides in a cytocentrifuge (Nordic, Tilburg). The cytocentrifuge preparations were air dried during 15 min and the location of the cells was marked by encircling with a glass pencil. The preparations were fixed in methanol (30 min, 4°C) and washed in PBS for 15 min. The slides were dried and incubated with 15/tl of a rabbit anti-TdT antiserum (Bethesda Research Laboratory, Gaithersburg, MD) in a moist chamber (30 min, RT). The slides were then washed in PBS for 15 min, dried and incubated with 15 /~1 FITC-conjugated goat anti-rabbit Ig antiserum (Bethesda Research Laboratory) in a moist chamber (30 rain, RT). The slides were washed again in PBS (15 rain), dried and mounted in glycerol/PBS (9:1) containing 1 mg phenylenediamine/ml to prevent fading of the fluorochromes (Johnson and Nogueira Araujo, 1981). A coverslip was sealed to the slide with paraffin wax with ceresin (BDH Chemicals, Poole, England).
Microscopes For evaluation of immunofluorescence and immunogold staining Zeiss and Leitz fluorescence microscopes were used. Both were equipped with phase contrast facilities and an adjustable aperture diaphragm in the epi-illumination system. The Zeiss microscope (Carl Zeiss, Oberkochen) was a Zeiss Universal !I, equipped with a III RS Fluoreszenz-Auflicht condenser and a 50 W HBO mercury lamp (Osram, Berlin) and two 63 × objectives (Carl Zeiss): neofluar 63/1.25 Oel Ph 3 and neofluar 63/1.25 Oel. Filter combination 19 (Carl Zeiss) was used for the evaluation of FITC and filter combination 14 (Carl Zeiss) for TRITC. The Leitz microscope (Ernst Leitz Wetzlar, Wetzlar) was a Leitz Orthoplan, equipped with a Leitz Ploemopak 2 illumination, a 100 W HBO mercury lamp (Osram), a Leitz Vario Orthomat camera and two 63 × objectives (Leitz): PL APO 63/1.40 Oel Phaco 4 and 63/1.30 Oel Fluoreszenz. Filter combination I2 (Leitz) was used for evaluation of FITC and filter combination N2 (Leitz) for TRITC. For evaluation of colloidal gold, we used in both microscopes a polarization filter combination, consisting of a polarizer for the excitation light, a beamsplitter and an analyzer that extinguished reflected light by having the same polarization as the excitation light (De Mey, 1983).
Results
Microscopes 63 x objectives with phase rings can be used for visualization of FITC and TRITC, but not for colloidal gold because of high background, probably due tO reflection and depolarization of the polarized light by the phase rings. Visualization of the colloidal gold was made possible by the use of 63 × objectives without phase
rings and by closing the aperture diaphragm of the epi-illumination system. With the phase contrast dia-illumination system, the gold particles were not detectable, but by epi-illumination with polarized light the gold-yellow scattered light was clearly visible. For routine evaluation of triple staining the Zeiss microscope was used.
Triple immunological staining In the BM samples tested the percentage TdT positive cells ranged from 0.5% to 10%. The triple staining revealed that the majority (more than 70%) of the TdT positive cells was positive for HLA-DR, but negative for the T cell marker 3A1 (Fig. 1). A minority (0.1-8%) of the TdT positive cells was positive for both H L A - D R and 3A1. Detailed studies of these cell populations will be published elsewhere.
Discussion
Colloidal gold particles are microscopically detectable by polarized light used in the same epi-illumination system as for fluorescence. Our results demonstrate that in triple immunological staining, colloidal gold particles can be used as a third label in combination with the fluorochromes FITC and TRITC. The triple staining of BM cells for 3A1, H L A - D R and TdT revealed that within the small TdT positive cell population subpopulations are present, e.g. a 3 A 1 - / H L A - D R + / T d T + subpopulation, and also a very small one of 3A1 + / H L A - D R + / T d T ÷ phenotype. Thus triple immunological staining can be a powerful tool in analyzing the heterogeneity of small cell populations in cell differentiation studies. Since we used 2 mouse McAb and a rabbit antiserum in the triple staining, it was sufficient to use only 1 conjugated McAb. However, where combination of 3 mouse McAb is required, it has hitherto been necessary to use at least 2 conjugated McAb or McAb differing in Ig (sub)class in combination with different conjugated antimouse Ig (sub)class antisera. A more elegant and less time-consuming solution is to use 3 differently conjugated McAb. Therefore it would be convenient if the McAb of the required specificities were not only available as FITC or T R I T C conjugates, but also as colloidal gold conjugates.
Acknowledgements
We gratefully acknowledge Prof. Dr. R. Benner's support, and thank Mrs. E.G.M. Cristen, A.A. van der Linde-Preesman and I.L.M. Tettero for their excellent technical assistance, Dr. J. De Mey from Janssen Pharmaceutica and Dr. J.H.W. Leuvering Fig. 1. Triple immunologicalstaining of BM cells for 3AI, HLA-DR and TdT. a: phase contrast; b: 3A1 positive cells (colloidalgold labelled); c: HLA-DR positivecells (TRITC labelled); d: TdT positive cells (FITC labelled). The 2 TdT positive cells are also positive for HLA-DR, but not for the T cell marker 3A1; they probably representprecursor B cells. Three cells are positivefor 3A1 and not for HLA-DR and TdT, probably representingnormal T lymphocytes.
f r o m O r g a n o n I n t e r n a t i o n a l B.V. a n d Dr. M. D e W a e l e for their advice, Mr. C . G . E n k e l a a r f r o m Z e i s s N e d e r l a n d for his t e c h n i c a l s u p p o r t , Mr. J. F e n g l e r for his p h o t o g r a p h i c assistance, a n d Mrs. C . J . M . M e i j e r i n k - C l e r k x for t y p i n g this manuscript.
N o t e Added in P r o o f T h e e v a l u a t i o n o f the c o l l o i d a l g o l d s t a i n i n g c a n be i m p r o v e d b y use of the Z e i s s objective Antiflex-Neofluar 63/1.25 Oel which contains a rotable quarter-wave plate.
References B0yum, A., 1968, Scand. J. Clin. Lab. Invest. 21, 77. Bradstock, K.F., G. Janossy, N. Tidman, E.S. Papageorgiou, H.G. Prentice, M. Willoughby and A.V. Hoffbrand, 1981, Leuk. Res. 5, 301. De Mey, J., 1983, in: Immunocytochemistry, Practical Application in Pathology and Biology, eds. J,M. Polak and S. Van Noorden (Wright PSG, Bristol) p. 82. Feller, A.C. and M.R. Parwaresch, 1983, J. Immunol. Methods 63, 273. Haynes, B.F., D.L. Mann, M.E. Hemler, J.A. Schroer, J.H. Shelhamer, G.S. Eisenbarth, J.L. Strominger, C.A. Thomas, H.S. Mostowski and A.S. Fa~uci, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 2914. Hijmans, W., J.J. Haaijman and H.R.E. Schuit, 1981, in: Immunological Techniques Applied to Aging Research, eds. W.H. Adler and A.A. Nordin (CRC Press, Boca Rato, FL) p. 141. Johnson, G.D. and G.M. de C. Nogueira Araujo, 1981, J. Immunol. Methods 43, 349. Lampson, L.A. and R. Levy, 1980, J. Immunol. 125, 293. Moir, D.J., A.K. Ghosh, Z. Abdulaziz, P.M. Knight and D.Y. Mason, 1983, Br. J. Haematol. 55, 395. Oi, V.T., A.N. Glazer and L. Stryer, 1982, J. Cell Biol. 93, 981. Ploem, J.S., 1967, Z. Wiss. Mikr. 68, 129. Rothbarth, Ph.H., H.J. Tanke, N.A.J. Mul, J.S. Ploem, J.F.G. Vliegenthart and R.E. Ballieux, 1978, J. Immunol. Methods 19, 101. Schuit, H.R.E. and W. Hijmans, 1980, Clin. Exp. Immunol. 41,567. Shapiro, H.M., 1983, Cytometry 3, 227. Titus, J.A., R. Haugland, S.O. Sharrow and D.M. Segal, 1982, J. Immunol. Methods 50, 193. Van Dongen, J.J.M., H. Hooijkaas, K. H~.hlen, K. Benne, W.M. Bitter, A.A. Van der Linde-Preesman, I.L.M. Tettero, M. Van de Rijn, J. Hilgers, G.E. Van Zanen and A. Hagemeijer, 1984, in: Minimal Residual Disease in Acute Leukemia, eds. B. L6wenberg and A. Hagenbeek (Martinus Nijhoff, Boston) p. 67.