Analysis of Tissue Imprints by Scanning Laser Cytometry

Reinhard Bollmann1, Gábor Méhes2

1 Institute of Pathology Bonn‐Duisdorf, Bonn, 2 University of Pécs, Pécs
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.22
DOI:  10.1002/0471142956.cy0722s26
Online Posting Date:  November, 2004
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Abstract

The analysis of DNA content and simultaneous surface or intracellular antigen expression is described for laser scanning cytometry. Measurements can be performed on microscope slides prepared immediately after dissection of native surgical specimens. These protocols will be useful for both diagnostic pathology and cell biology laboratories.

Keywords: Ploidy; DNA; immunofluorescence; laser scanning cytometry; breast cancer

     
 
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Table of Contents

  • Basic Protocol 1: Direct Immunofluorescence Labeling and DNA Staining of Tissue Imprints for Analysis by Scanning Laser Cytometry
  • Alternate Protocol 1: Indirect Immunofluorescence Labeling and DNA Staining of Tissue Imprints for Analysis by Scanning Laser Cytometry
  • Support Protocol 1: Preparation of a Tissue Imprint
  • Support Protocol 2: Preparation of a Tissue Imprint Using a Cytological Device
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Immunofluorescence Labeling and DNA Staining of Tissue Imprints for Analysis by Scanning Laser Cytometry

  Materials
  • Microscope slide containing tumor tissue imprint (see protocol 3 and protocol 4), smear, or cytospin preparation (unit 5.2)
  • Acetone, ice cold (−20°C)
  • 4% (w/v) BSA in PBS ( appendix 2A) or commercial blocking solution (e.g., Protein Block; BioGenex Laboratories)
  • FITC‐conjugated mouse CD45 antibody (Dako), diluted 1:10 with PBS
  • FITC‐conjugated mouse IgG 1 isotype antibody (Dako), diluted 1:10 with PBS
  • PI/RNase solution (see recipe)
  • Glycerol
  • Diamond pencil
  • Humidified chamber: ∼15 × 30–cm stainless steel or plastic container with lid (for up to ten slides), containing one or two paper towels moistened with distilled water
  • 37°C incubator
  • Coverslips (10 × 10 mm or cut to fit area containing cells)
  • Laser scanning cytometer (LSC; e.g., CompuCyte) equipped with argon (Ar) and helium/neon (HeNe) lasers; standard filter settings for the measurement of FITC and PI fluorescence

Alternate Protocol 1: Indirect Immunofluorescence Labeling and DNA Staining of Tissue Imprints for Analysis by Scanning Laser Cytometry

  • Unconjugated mouse antibody, such as:
    • NCL‐ER6F11 (Novocastra Labs) for estrogen receptor, diluted 1:10 in PBS ( appendix 2A)
    • NCL‐PGR (Novocastra Labs) for progesterone receptor, diluted 1:10 in PBS anti‐Ki‐67 (Linaris) or NCL‐Ki67‐MM1 (Novocastra Labs) for proliferating cells, diluted 1:20 in PBS
  • Unconjugated isotype control IgG (e.g., Dako), diluted as for antibody of interest
  • PBS ( appendix 2A)
  • Biotinylated anti‐mouse antibody (e.g., BioGenex Laboratories), diluted in PBS according to manufacturer's instructions
  • FITC‐conjugated streptavidin (e.g., Dako), diluted in PBS according to manufacturer's instructions

Support Protocol 1: Preparation of a Tissue Imprint

  Materials
  • Freshly prepared solid tumor sample (prevent tissue from drying out)
  • Positively charged microscope slides
  • Lint‐free tissues
  • Scalpel, sharp razor blade, previously used microtome blade, or other dissection instrument
  • Filter paper
  • Grease pencil (e.g., ImmEdge; Vector Laboratories)
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Figures

Videos

Literature Cited

Literature Cited
   Bergers, E., Baak, J.P.A., van Diest, P.J., Willig, A.J.P., Los, J., Peterse, J.L., Ruitenberg, H.M., Schapers, R.F.M., Somsen, J.G., van Beek, M.W.P.M., Bellot, S.M., Fijnheer, J., and van Gorp, L.H.M. 1997. Prognostic value of DNA ploidy using flow cytometry in 1301 breast cancer patients: Result of the prospective multicenter morphometric mammary carcinoma project. Mod. Pathol. 10:762–768.
   Bollmann, R., Torka, R., Schmitz, J., Bollmann, M., and hes, G. 2002. Determination of ploidy and steroid receptor status in breast cancer by laser scanning cytometry. Cytometry 50:201–215.
   Fitzgibbons, P.L., Page, D.L., Weaver, D., Thor, A.D., Allred, C.D., Clark, G.M., Ruby, S.G., O'Malley, F., Simpson, J.F., Connolly, J.L., Hayes, D.F., Edge, S.B., Lichter, A., and Schnitt, S.J. 2000. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement. Arch. Pathol. Lab. Med. 124:966–978.
   Gorczyca, W., Darzynkiewicz, Z., and Melamed, M.R. 1997. Laser scanning cytometry in the pathology of solid tumors. Acta Cytol. 41:98–108.
   Kamentsky, L.A. and Kamentsky, L.D. 1991. Microscope based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381–387.
   Nicholson, R.I., McClelland, R.A., and Gee, J.M. 1995. Steroid hormone receptors and their clinical significance in cancer. Am. J. Clin. Pathol. 48:890–895.
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