Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification

Peter F. Ambros1, Gábor Méhes2

1 Children's Cancer Research Institute (CCRI), St. Anna Children's Hospital, Vienna, 2 University of Pécs, Pécs
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.13
DOI:  10.1002/0471142956.cy0813s26
Online Posting Date:  November, 2004
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Abstract

Fluorescence‐microscope based methods are presented that allow automatic selection and quantification of immunophenotyped cells and sequential FISH analysis to facilitate molecular cytogenetic analysis of single cells and very small cell populations. The protocols in this unit are particularly useful for studying the biological and genetic makeup of disseminated tumor cells or other rare cells.

Keywords: fluorescence microscopy; immunofluorescence labeling; FISH; cytogenetic analysis

     
 
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Table of Contents

  • Basic Protocol 1: Immunofluorescence Detection and Characterization of Rare Cells
  • Basic Protocol 2: FISH After Immunofluorescence Staining
  • Alternate Protocol 1: Sequential FISH Using Directly Labeled DNA Probes Following Immunofluorescent Analysis
  • Alternate Protocol 2: Multiple Sequential FISH Following Immunolabeling
  • Support Protocol 1: Mononuclear Cell Preparation for Sequential Immunolabeling
  • Support Protocol 2: Automated Fluorescence Image Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunofluorescence Detection and Characterization of Rare Cells

  Materials
  • Cytological specimen: cytospin preparation (see, e.g., protocol 5), smear, or touch slide
  • 3.7% (v/v) formaldehyde in PBS (see appendix 2A for PBS)
  • Phosphate‐buffered saline (PBS), pH 7.0 ( appendix 2A)
  • 2% and 4% (w/v) bovine serum albumin (BSA; Sigma) in PBS (see appendix 2A); filter sterilize before use
  • Primary antibody: e.g., anti‐cytokeratin (MNF 116, available from Dako) or anti‐GD2 (available from Dr. R. Reisfeld, Scripps Clinic, La Jolla, Calif.)
  • Fluorescently labeled secondary antibody directed against species in which primary antibody was raised: e.g., FITC‐conjugated anti‐mouse
  • 0.2 µg/ml DAPI (Sigma) in PBS (see appendix 2A)
  • Moist chamber: e.g., rectangular plastic box with lid, containing paper towels moistened with deionized H 2O, accommodating up to 10 slides
  • Glass coverslips
  • Antifade mounting medium containing DAPI (e.g., Vectashield, Vector Laboratories)
  • Fluorescence microscope (e.g., Axioplan, Zeiss)
  • Digital image‐analysis workstation (optional; see protocol 6)

Basic Protocol 2: FISH After Immunofluorescence Staining

  Materials
  • Immunofluorescently labeled slides (see protocol 1)
  • Phosphate‐buffered saline (PBS), pH 7.0 ( appendix 2A)
  • 0.05% pepsin solution (see recipe)
  • 70%, 96%, and 100% ethanol
  • Hybridization mix (see recipe) containing biotin‐ or digoxigenin‐labeled DNA probes (ideally BAC, PAC, YAC, or cosmid clones or repetitive sequences)
  • Rubber cement
  • 2× and 4× SSC ( appendix 2A)
  • Post‐hybridization wash solution: e.g., 50% (v/v) formamide in 2× SSC, prewarmed to 42°C
  • 2% and 4% (w/v) bovine serum albumin (BSA; Sigma) in PBS (see appendix 2A); filter sterilize before use
  • Primary antibodies: e.g., mouse anti‐biotin (Dako), FITC‐conjugated sheep anti‐digoxigenin (e.g., Boehringer Mannheim)
  • Secondary antibodies: fluorochrome‐conjugated antibodies directed against species from which primary antibody was derived (e.g., TRITC‐conjugated rabbit anti‐mouse, FITC‐conjugated rabbit anti‐sheep (e.g., Dako))
  • 0.1% (v/v) Tween 20 in 4× SSC (see appendix 2A for SSC)
  • 3.7% (v/v) formaldehyde in PBS (see appendix 2A for PBS)
  • Antifade mounting medium containing DAPI (e.g., Vectashield, Vector Laboratories)
  • Coplin jars
  • Moist chamber: e.g., rectangular plastic box with lid, containing paper towels moistened with deionized H 2O, accommodating up to 10 slides
  • Coverslips

Alternate Protocol 1: Sequential FISH Using Directly Labeled DNA Probes Following Immunofluorescent Analysis

  • DNA probes with direct fluorochrome labeling (purchase from Qbiogene, use LSI probes from Vysis, or prepare home‐made probes as described in unit 8.3) ; store DNA probes at –20°C; store ready‐to‐use probe mixtures at 4°C

Alternate Protocol 2: Multiple Sequential FISH Following Immunolabeling

  • 65% (v/v) formamide in 2× SSC (see appendix 2A for SSC)

Support Protocol 1: Mononuclear Cell Preparation for Sequential Immunolabeling

  Materials
  • Lymphoprep (Nycomed)
  • Phosphate‐buffered saline (PBS), pH 7.0 ( appendix 2A)
  • Erythrolysis buffer (see recipe)
  • RPMI 1640 medium containing 10% FBS ( appendix 3B)
  • 240‐mm2 Hettich Cytospin cytocentrifuge
  • 15‐ml conical polypropylene tubes
  • Benchtop centrifuge
  • Glass slides (e.g., HistoBond from Paul Marienfeld, Heidelberg, Germany)
  • Vacuum aspirator
  • Additional reagents and equipment for counting cells using a Coulter counter ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Ambros, P.F., Méhes, G., Hattinger, C.M., Ambros, I.M., Luegmayr, A., Ladenstein, R., and Gadner, H. 2001. Unequivocal identification of tumor cells in the bone marrow by combining immunological and genetic approaches: Functional and prognostic information. Leukemia 15:275‐277.
  The help of Andrea Luegmayr, Elisabeth Vitasek, and Rita Narath is greatly acknowledged. This work was supported by the Children's Cancer Research Institute (CCRI).
   Ambros, P.F., Méhes, G., Ambros, I.M., and Ladenstein, R. 2003. Disseminated tumor cells in the bone marrow: Chances and consequences of microscopical detection methods. Cancer Lett. 197:29‐34.
   Hopman, A.H., Voorter, C.E., and Ramaekers, F.C. 1994. Detection of genomic changes in cancer by in situ hybridization. Mol. Biol. Rep. 19:31‐44.
   Knuutila, S. 1993. Simultaneous detection of immunophenotype and genome by the MAC method. J. Histochem. Cytochem. 41:1715‐1716.
   Méhes, G., Lörch, T., and Ambros, P.F. 2000. Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis. Cytometry 42:357‐362.
   Méhes, G., Luegmayr, A., Hattinger, C.M., Lörch, T., Ambros, I.M., Gadner, H., and Ambros, P.F. 2001. Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow. Clin. Cancer Res. 7:1969‐1975.
   Nylund S.J., Wessman, M., and Larramendy, M.L. 1994. Analysis of genotype and phenotype on the same interphase or mitotic cell: A manual of MAC (morphology antibody chromosomes) methodology. Cancer Genet. Cytogenet. 72:1‐15.
   Speel, E.J.M., Herbergs, J., Ramaekers, F.C.S., and Hopman, A.H.N. 1994. Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42:961‐966.
   Strehl, S. and Ambros, P.F. 1993. Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet. Cell Genet. 63:24‐28.
   Weber‐Matthiesen, K., Deerberg, J., Müller‐Hermelink, A., Schlegelberger, B., and Grote, W. 1993. Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method. Cytogenet. Cell Genet. 63:123‐125
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