Combination DNA/RNA Fish and Immunophenotyping

Roeland W. Dirks1

1 Leiden University Medical Centre, Leiden, The Netherlands
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.7
DOI:  10.1002/0471142956.cy0807s06
Online Posting Date:  May, 2001
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Abstract

This unit presents methods for combining immunophenotyping with DNA/RNA FISH. The approach is used in so‐called genotype/phenotype analysis to identify chromosomal aberrations in sub‐populations of cells present in heterogenous populations. Combining RNA and DNA detection with identification of cellular proteins is quite difficult. This series of protocols is provided to enable the successful application of the combination of these techniques.

     
 
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Table of Contents

  • Basic Protocol 1: Combined Detection of DNA and Protein
  • Basic Protocol 2: Combined Detection of mRNA and Protein
  • Alternate Protocol 1: Detection of a Protein Followed by Detection of mRNA Species
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Combined Detection of DNA and Protein

  Materials
  • 2% and 4% formaldehyde fixative (see recipe)
  • Cell suspension: 6 × 106 cells/ml in 1× PBS (see unit 8.2 for PBS)
  • 1× PBS (unit 8.2) containing 1% (w/v) BSA
  • 1:1 (v/v) ethanol/ether
  • Tris‐buffered saline (TBS; appendix 2A)
  • TBS blocking buffer (see recipe)
  • Primary antibodies: dilute cell type–specific antibody according to manufacturer's instructions in TBS blocking buffer (see recipe for TBS blocking buffer) and microcentrifuge 3 min at 10,000 × g
  • Secondary antibody: dilute hapten‐ (e.g., biotin)‐labeled antibody in TBS blocking buffer according to manufacturer's instructions (see recipe for TBS blocking buffer) and microcentrifuge 3 min at 10,000 × g
  • Phosphate‐buffered saline (1× PBS; unit 8.2)
  • 0.1% pepsin (Sigma) in 0.01 M HCl (pH 2.0), 37°C
  • 70%, 90%, and 100% ethanol
  • 10‐ml polypropylene centrifuge tubes
  • Mechanical shaker
  • Dust‐free Kimwipes
  • Cytospin 2 tabletop cytocentrifuge (Shandon/Lipshaw)
  • Microscope slides
  • 24 × 60–mm and 50 × 60–mm coverslips
  • Schiefferdecker staining jars (e.g., Fisher)
  • Moist chamber: 1‐liter beaker containing paper towels moistened with water, covered with aluminum foil
  • Additional reagents and equipment for cell counting ( appendix 3A), DNA FISH (unit 8.3), and probe visualization (unit 8.5)

Basic Protocol 2: Combined Detection of mRNA and Protein

  Materials
  • Cells of interest
  • Culture medium (e.g., complete DMEM/10% FBS; see appendix 2A) without phenol red
  • Phosphate‐buffered saline (1× PBS; unit 8.2)
  • Formaldehyde/acetic acid fixative (see recipe)
  • 70%, 90%, and 100% ethanol
  • 0.1% pepsin (Sigma) in 0.01 M HCl (pH 2.0), 37°C
  • 4% formaldehyde fixative (see recipe; optional)
  • Hybridization mix (see recipe) containing nick‐translated probe and (optionally) without probe
  • 50% formamide/2× SSC (see appendix 2A for SSC), pH 7.0 (37°C)
  • 2× SSC ( appendix 2A)
  • Tris‐buffered saline (TBS; appendix 2A)
  • Primary antibodies: against hapten used to label probe and against cell‐specific protein of interest, obtained from different animal species
  • TBS blocking buffer (see recipe)
  • Secondary antibodies conjugated to fluorochrome or horseradish peroxidase
  • Mounting medium (e.g., Vectashield from Vector Labs)
  • Microscope slides
  • 180°C oven
  • Sterile 15 × 15–cm petri dishes
  • 37°C humidified 5% CO 2 incubator
  • Hettich‐Universal tabletop cytocentrifuge with no. 1323 rotor and compatible buckets (no. 1266, no. 1271, or no. 1276) for depositing cells on slides (Hettich‐Zentrifugen)
  • Staining jars
  • 18 × 18–mm and 24 × 60–mm coverslips
  • Moist chamber: 1‐liter beaker containing paper towels moistened with 50% formamide, sealed with Parafilm and aluminum foil
  • Metal plate heated to 80°C in oven
  • 37°C shaking water bath
  • Slide boxes
  • Fluorescence microscope equipped with appropriate filter sets for red, green, and blue excitation, high‐NA Plan Apo lenses, and HBO 100‐W mercury arc lamp (unit 2.4)
  • Camera system with ISO 640 color slide film (e.g., 3M) or CCD camera system and computer running image analysis system and Adobe Photoshop (unit 2.5)
  • Additional reagents and equipment for immunocytochemical detection (unit 8.4)
CAUTION: Formaldehyde and formamide are hazardous and should be handled with care (see manufacturer's instructions).NOTE: All buffer solutions should be autoclaved.

Alternate Protocol 1: Detection of a Protein Followed by Detection of mRNA Species

  • PBS/DEPC (see recipe)
  • Blocking reagent for nucleic acid hybridization (Boehringer Mannheim)
  • RNasin ribonuclease inhibitor (Promega)
  • 2% formaldehyde fixative (see recipe)
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Figures

Videos

Literature Cited

   Dirks, R.W., Van de Rijke, F.M., Fujishita, S., Van der Ploeg, M., and Raap, A.K. 1993. Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104:1187‐1197.
   Dirks, R.W., de Pauw, E.S.D., and Raap, A.K. 1997. Splicing factors associate with nuclear HCMV‐IE transcripts after transcriptional activation of the gene, but dissociate upon transcription inhibition: Evidence for a dynamic organization of splicing factors. J. Cell Sci. 110:515‐522.
   Hessel, H., Mittermüller, J., Zitzelsberger, H., Weier, H.‐U., and Bauchinger, M. 1996. Combined immunophenotyping and FISH with sex chromosome‐specific DNA probes for the detection of chimerism in epidermal Langerhans cells after sex‐mismatched bone marrow transplantation. Histochem. Cell Biol. 106:481‐485.
   Höltke, H.J. and Kessler, C. 1990. Non‐radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (Dig): Hybridization and ELISA‐based detection. Nucleic Acids Res. 18:5843‐5851.
   Huang, S. and Spector, D.L. 1996. Intron‐dependent recruitment of pre‐mRNA splicing factors to sites of transcription. J. Cell Biol. 133:719‐732.
   Lawrence, J.B., Singer, R.H., and Marselle, LM. 1989. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57:493‐502.
   Litle, V.R., Lockett, S.J., and Pallavicini, M.G. 1996. Genotype/phenotype analyses of low frequency tumor cells using computerized image microscopy. Cytometry 23:344‐349.
   Raap, A.K., Van der Corput, M.P.C., Vervenne, R.A.W., Van Gijlswijk, R.P.M., and Tanke, H.J. 1995. Ultra‐sensitive FISH using peroxidase‐mediated deposition of biotin‐ or fluorochrome tyramides. Hum. Mol. Genet. 4:529‐534.
   Singer, R.H., Lawrence, J.B., and Villnave, C.A. 1986. Optimization of in situ hybridization using isotopic and non‐isotopic detection methods. BioTechniques 4:230‐250.
   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.
   Weber‐Matthiesen, K., Pressl, S., Schlegelberger, B., and Grote, W. 1993. Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method. Leukemia 74:646‐649.
   Xing, Y., Johnson, C.V., Moen, P.T., McNeil, J.A., and Lawrence, J.B. 1995. Nonrandom gene organization: structural arrangements of specific pre‐mRNA transcription and splicing with SC‐35 domains. J. Cell Biol. 131:1635‐1647.
Key References
   Bakkus, M.H.C., Brakel van Peer, K.M.J., Adriaansen, H.J., Wierenga‐Wolf, A.F., Van den Akker, T.W., Dicke‐Evinger, M.J., and Benner, R. 1989. Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers. Oncogene 4:1255‐1262.
  Early report of a method that allows the study of gene expression in phenotypically defined cell populations.
   Dirks et al., 1993. See above.
  Describes an optimized protocol for RNA FISH on cultured cells.
   Harper, S.J., Pringle, J.H., Allen, A.C., Layward, L., Feehally, J., and Lauder, I. 1992. Simultaneous in situ hybridization of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin wax embedding sections. J. Clin. Pathol. 45:114‐119.
  Describes a method for simultaneous detection of a native mRNA by FISH and a cytoplasmic antigen by immunofluorescence in routine pathology specimens.
   Raap, A.K., Van de Rijke, F.M., Dirks, R.W., Sol, C.J., Boom, R., and Van der Ploeg, M. 1991. Bicolor fluorescence in situ hybridization to intron and exon mRNA species. Exp. Cell Res. 197:319‐322.
  Describes the combined detection of an RNA species with its cognate protein and with incorporated BrdU.
   Singer et al., 1986. See above.
  Contains a detailed evaluation of hybridization parameters for RNA ISH.
   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.
  First description of a technique for double‐target FISH and simultaneous immunological staining of a cell‐surface antigen.
   Van den Berg, H., Vossen, J.M., Van den Bergh, R.L., Bayer, J., and Van Tol, M.J.D. 1991. Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two‐color immunofluorescence. Lab Invest. 64:623‐628.
  Describes a technique for simultaneous detection of the Y chromosome and differentiation‐specific membrane antigens on peripheral‐blood mononuclear cells.
   Weber‐Matthiesen, K., Winkemann, M., Muller‐Hermelink, A., Schlegelberger, B., and Grote, W. 1992. Simultaneous fluorescence immunophenotyping and interphase cytogenetics: A contribution to the characterization of tumor cells. J. Histochem. Cytochem. 40:171‐175.
  Provides a method that combines immunofluorescence immunophenotyping and FISH using centromere‐specific cDNA probes on cytospin preparations.
   Weber‐Matthiesen et al., 1993. See above.
  First description of a technique combining interphase cytogenetics and immunophenotyping on cryostat sections.
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