Immunocytochemical Detection

J. Wiegant1

1 Leiden University, Leiden
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.4
DOI:  10.1002/0471142956.cy0804s04
Online Posting Date:  May, 2001
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Abstract

This unit provides detailed descriptions for direct and indirect in situ hybridization methods. Protocols are designed for bright‐field microscopy using a precipitating chromogenic substrate and for immunofluorescence detection using a hapten‐labeled probe. Several alternative procedures are provided together with an excellent discussion of the strategy used for selecting a detection method.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Alkaline Phosphatase/NBT‐BCIP Reaction for BfISH Using Digoxigenin‐, Biotin‐, or FITC‐labeled Hybridization Probes
  • Basic Protocol 2: Peroxidase/DAB Reaction for BfISH Using Digoxigenin‐, Biotin‐ or FITC‐labeled Hybridization Probes
  • Basic Protocol 3: Single‐color Detection for FISH Using Biotin‐Labeled Hybridization Probes
  • Alternate Protocol 1: Single‐color Detection for FISH Using Digoxigenin‐labeled Hybridization Probes
  • Alternate Protocol 2: Single‐color Detection for FISH Using FITC‐labeled Hybridization Probes
  • Basic Protocol 4: Dual‐color Detection for FISH Using Biotin‐ and Digoxiginin‐Labeled Hybridization Probes
  • Basic Protocol 5: Triple‐color Detection for Fish
  • Basic Protocol 6: Tyramide Signal Amplification
  • Support Protocol 1: Handling of Preparations and Reagents for Immunocytochemical Detection
  • Support Protocol 2: Embedding of Preparations for Evaluation by Fluorescence Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Alkaline Phosphatase/NBT‐BCIP Reaction for BfISH Using Digoxigenin‐, Biotin‐, or FITC‐labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with fluorescein (FITC)–, digoxigenin‐, or biotin‐labeled probes (unit 8.3)
  • First antibody for high sensitivity (see Table 8.4.1)
  • TNT buffer (see recipe)
  • Second antibody for high sensitivity (see Table 8.4.1)
  • Third antibody for high sensitivity (see Table 8.4.1)
  • Alkaline phosphatase (AP)–conjugated antibody for low sensitivity (select one): 1:1000 AP‐sheep‐anti‐digoxigenin (Boehringer Mannheim), 1:200 AP‐streptavidin (Vector Labs), or 1:500 AP‐mouse‐anti‐FITC (Dako)
  • Alkaline phosphatase (AP) buffer (see recipe)
  • Alkaline phosphatase (AP) substrate solution (see recipe)
  • Nuclear fast red (Sigma)
  • Aquamount (BDH) or equivalent mounting medium for AP reaction
  • 24 × 50–mm cover slips
  • Moist chamber: 1‐liter beaker containing paper towels moistened with TNT buffer (see recipe), covered with aluminum foil
  • Schiefferdecker or Coplin jars (Fisher Scientific)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9)

Basic Protocol 2: Peroxidase/DAB Reaction for BfISH Using Digoxigenin‐, Biotin‐ or FITC‐labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with fluorescein (FITC)–, digoxigenin‐, or biotin‐labeled probes (unit 8.3)
  • First antibody for high sensitivity (see Table 8.4.2)
  • TNT buffer (see recipe)
  • Second antibody for high sensitivity (see Table 8.4.2)
  • Third antibody for high sensitivity (see Table 8.4.2)
  • Horseradish peroxidase (HRP)–conjugated antibody for low sensitivity (select one): 1:100 HRP‐sheep‐anti‐digoxigenin (Boehringer Mannheim), 1:100 HRP‐streptavidin (Vector Labs), or 1:500 HRP‐sheep‐anti‐FITC (NEN Life Sciences)
  • DAB substrate solution (see recipe)
  • Hematoxylin
  • 70%, 90%, and 100% ethanol
  • 1:1 (v/v) ethanol/xylene
  • 100% xylene
  • Fluoromount mounting medium (BDH)
  • Schiefferdecker or Coplin jars (Fisher Scientific)
  • 24 × 50–mm coverslips
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9)

Basic Protocol 3: Single‐color Detection for FISH Using Biotin‐Labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with a biotinylated probe (unit 8.3)
  • 1:100 fluorochrome‐streptavidin (Vector Labs)
  • TNT buffer (see recipe)
  • 1:100 biotin‐goat‐anti‐streptavidin (Vector Labs)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9) and for embedding the preparation for fluorescence microscopy (see protocol 10)

Alternate Protocol 1: Single‐color Detection for FISH Using Digoxigenin‐labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with a digoxigenin‐labeled probe (unit 8.3)
  • 1:100 fluorochrome‐sheep‐anti‐digoxigenin (Boehringer Mannheim)
  • TNT buffer (see recipe)
  • 1:500 mouse anti‐digoxin (Sigma)
  • 1:200 mouse anti‐digoxigenin (Boehringer Mannheim)
  • 1:1000 fluorochrome‐rabbit‐anti‐mouse (Sigma)
  • 1:250 fluorochrome‐goat‐anti‐rabbit (Vector Labs)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9) and for embedding the preparation for fluorescence microscopy (see protocol 10)

Alternate Protocol 2: Single‐color Detection for FISH Using FITC‐labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with a FITC‐labeled probe (unit 8.3)
  • 1:250 rabbit anti‐FITC (Dako)
  • TNT buffer (see recipe)
  • 1:1000 FITC‐goat‐anti‐rabbit (Vector Labs)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9) and for embedding the preparation for fluorescence microscopy (see protocol 10)

Basic Protocol 4: Dual‐color Detection for FISH Using Biotin‐ and Digoxiginin‐Labeled Hybridization Probes

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with one biotin‐labeled and one digoxigenin‐labeled probe (unit 8.3)
  • 1:100 FITC‐sheep‐anti‐digoxigenin/1:100 Texas red–streptavidin (Boehringer Mannheim and Vector Labs, respectively)
  • TNT buffer (see recipe)
  • 1:200 mouse anti‐digoxigenin/1:100 Texas red–streptavidin (Boehringer Mannheim and Vector Labs, respectively)
  • 1:1000 FITC‐rabbit‐anti‐mouse/1:100 biotin‐goat‐anti‐streptavidin (Sigma and Vector Labs, respectively)
  • 1:250 FITC‐goat‐anti‐rabbit/1:100 Texas red–streptavidin (Vector Labs)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9) and for embedding the preparation for fluorescence microscopy (see protocol 10)

Basic Protocol 5: Triple‐color Detection for Fish

  Materials
  • ISH‐labeled preparation: chromosomes hybridized with one digoxigenin‐labeled probe, one biotinylated probe, and one FITC‐labeled probe (unit 8.3)
  • 1:100 aminomethyl coumarin acetic acid (AMCA)‐sheep‐anti‐digoxigenin/1:100 Texas red–streptavidin (Boehringer Mannheim and Vector Labs, respectively)
  • TNT buffer (see recipe)
  • 1:500 mouse anti‐digoxin/1:250 rabbit anti‐FITC/1:100 Texas red–streptavidin (Sigma, Dako, and Vector Labs, respectively)
  • 1:100 digoxigenin‐sheep‐anti‐mouse/1:250 FITC‐goat‐anti‐rabbit/1:100 biotin‐goat‐anti‐streptavidin (Boehringer Mannheim, Vector Labs, Vector Labs, respectively)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9) and for embedding the preparation for fluorescence microscopy (see protocol 10)

Basic Protocol 6: Tyramide Signal Amplification

  Materials
  • Tyramide solution (see recipe)
  • HRP‐labeled preparation (low‐sensitivity procedure; see protocol 2, steps , , and )
  • TNT buffer (see recipe)
  • 1:500 AP‐, HRP‐, or fluorochrome‐streptavidin (Vector Labs)
  • Additional reagents and equipment for handling of ISH samples and immunoreagents (see protocol 9), embedding the preparation for fluorescence microscopy (see protocol 10), and for alkaline phosphatase (see protocol 1) and peroxidase (see protocol 2) staining

Support Protocol 1: Handling of Preparations and Reagents for Immunocytochemical Detection

  Materials
  • ISH‐labeled preparation: chromosomes hybridized by appropriate probe (unit 8.3)
  • TNT buffer (see recipe)
  • TNB buffer (see recipe)
  • Immunoreagents (see recipe)
  • Schiefferdecker or Coplin jars
  • 24 × 50–mm glass coverslips
  • Moist chamber: 1‐liter beaker containing paper towels moistened with TNT buffer (see recipe), covered with aluminum foil

Support Protocol 2: Embedding of Preparations for Evaluation by Fluorescence Microscopy

  Materials
  • Fluorescently labeled ISH preparation (refer to appropriate labeling protocol), washed in TNT buffer
  • 70%, 90%, and 100% ethanol
  • Vectashield with or without DNA counterstain (see recipe)
  • 24 × 50–mm glass coverslips
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Figures

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Literature Cited

Literature Cited
   Florijn, R.J., Slats, J., Tanke, H.J., and Raap, A.K. 1995. Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177‐182.
   Kerstens, H.M.J., Poddighe, P.J., and Hanselaar, A.G.J.M. 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43:347‐352.
   Linsenmayer, T.F., Fitch, J.M., and Schmid, T.M. 1988. Multiple‐reaction cycling: A method for enhancement of the immunocytochemical signal of monoclonal antibodies. J. Histochem. Cytochem. 36:1075‐1078.
   Nederlof, P.M., Robinson, D., Abuknesha, R., Wiegant, J., Hopman, A.H.N., Tanke, H.J., and Raap, A.K. 1989. Three‐color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry. 10:20‐27.
   Pinkel, D., Straume, T., and Gray, J. 1986. Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. U.S.A. 83:2934‐2938.
   Raap, A.K., van de Corput, M.P.C., Vervenne, R.A.W., van Gijlswijk, R.P.M., Tanke, H.J., and Wiegant, J. 1995. Ultra‐sensitive FISH using peroxidase‐mediated deposition of biotin‐ or fluorochrome‐tyramides. Hum. Mol. Genet. 4:529‐534.
   Speel, E.J.M., Jansen, M.P.H.M., Ramaekers, F.C.S., and Hopman, A.H.N. 1994. A novel triple‐color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J. Histochem. Cytochem. 42:1299‐1307.
   Van Gijlswijk, R.P.M., Wiegant, J., Raap, A.K., and Tanke, H.J. 1996a. Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulphate and polyvinyl alcohol. J. Histochem. Cytochem. 44:389‐392.
   Van Gijlswijk, R.P.M., Wiegant, J., Vervenne, R., Lasan, R., Tanke, H.J., and Raap, A.K. 1996b. Horseradish peroxidase‐labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome‐specific repeat sequences. Cytogenet. Cell Genet. 75:258‐262.
   Wiegant, J., Ried, T., Nederlof, P.M., van der Ploeg, M., Tanke, H.J., and Raap, A.K. 1991. In situ hybridization with fluoresceinated DNA. Nucl. Acids Res.. 19:3237‐3241.
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