Hybridization Histochemistry of Neural Transcripts

W. Scott Young1, June Song1, Éva Mezey2

1 Section on Neural Gene Expression, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, 2 Adult Stem Cell Section, National Institute of Dental and Craniofacial Research, Nationals Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.3
DOI:  10.1002/cpns.9
Online Posting Date:  April, 2016
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Abstract

Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (or in situ hybridization) permits localization of these transcripts with cellular resolution or better. Furthermore, the relative amounts of transcripts detected in different tissues or in the same tissues in different states (e.g., physiological or developmental) may be quantified.

This unit describes hybridization histochemical techniques using either oligodeoxynucleotide probes (see Basic Protocols 1 and 2, Alternate Protocol 1) or RNA probes (riboprobes; see Basic Protocols 3 and 5). These methods include a more recent approach using commercially available sets of oligodeoxynucleotide pairs for colorimetric and fluorescent detection (see Basic Protocol 2), as well as a method for detection of the Y chromosome using either mouse or human riboprobes (see Basic Protocol 5). Additional methods include colorimetric detection (see Basic Protocol 4) and tyramide signal amplification (TSA) of digoxigenin‐labeled probes (see Alternate Protocol 2), and autoradiographic detection of radiolabeled probes (see Basic Protocol 6). Finally, methods are provided for labeling oligodeoxynucleotide (see Support Protocol 1) and RNA (see Support Protocol 2) probes, and verifying the probes by northern analysis (see Support Protocol 3). © 2016 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Basic Protocol 1: Hybridization Histochemistry with Oligodeoxynucleotide Probes
  • Basic Protocol 2: Hybridization Histochemistry for Detection of a Single Transcript Using Sets of Oligodeoxynucleotide Probe Pairs
  • Alternate Protocol 1: Hybridization Histochemistry for Simultaneous Detection of Two Transcripts Using Sets of Oligodeoxynucleotide Probe Pairs
  • Basic Protocol 3: Hybridization Histochemistry with RNA Probes
  • Basic Protocol 4: Detection of Digoxigenin‐Labeled Probes Using AP‐Conjugated Antibodies
  • Alternate Protocol 2: Detection of Digoxigenin‐Labeled Probes Using Tyramide Signal Amplification (TSA)
  • Basic Protocol 5: Detection of Y Chromosome DNA Using Riboprobes
  • Basic Protocol 6: Detection of Radiolabeled Probes
  • Support Protocol 1: Preparation of Oligodeoxynucleotide Probes for Hybridization Histochemistry
  • Support Protocol 2: Preparation of RNA Probes for Hybridization Histochemistry
  • Support Protocol 3: Northern Analysis Using Oligodeoxynucleotide Probes and Riboprobes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Hybridization Histochemistry with Oligodeoxynucleotide Probes

  Materials
  • 50% (v/v) formamide/4× SSPE ( appendix 2A)
  • 12‐μm cryostat tissue sections mounted on Superfrost Plus slides (Fisher) and defatted (unit 1.1)
  • Hybridization solution (see recipe)
  • 35S‐ or digoxigenin‐labeled oligodeoxynucleotide probe (see protocol 9)
  • 1× SSPE/1 mM dithiothreitol (DTT)
  • 70% (v/v) ethanol
  • Sterile Bio‐Assay dishes (Nunc; 245 × 245 × 30 mm)
  • Whatman 3MM chromatography paper
  • Glass coverslips
  • Staining dishes with slide racks
  • 55°C water bath
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps.

Basic Protocol 2: Hybridization Histochemistry for Detection of a Single Transcript Using Sets of Oligodeoxynucleotide Probe Pairs

  Materials
  • 37% (w/v) formaldehyde
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 16‐μm cryostat tissue sections (unit 1.1) mounted on Superfrost Plus slides (Fisher)
  • Quantigene ViewRNA ISH Tissue 2‐plex Assay kit (Affymetrix) including:
  • Wash Buffer Components 1 and 2
  • Probe Set Diluent QT
  • Protease QF
  • PreAmplifier Mix QT
  • Amplifier Mix QT
  • Label Probe Diluent QF
  • AP Enhancer Solution
  • Fast Red tablets
  • Naphthol Buffer
  • Blue Buffer
  • Blue Reagents 1‐3
  • Label Probe 1‐AP
  • Label Probe 6‐AP
  • 50%, 70%, and 100% (v/v) ethanol
  • Custom‐designed probe set(s) (Affymetrix)
  • Bacterial Type XXIV Proteinase (Sigma‐Aldrich, cat. no. P8038)
  • 0.3 μg/ml 4′,6‐diamidine‐2′‐phenylindole dihydrochloride (DAPI)
  • 0.1 M Tris·Cl, pH 7.4 ( appendix 2A)
  • Coplin jars
  • Staining dishes with slide racks
  • Oven at 60°C
  • Hydrophobic barrier pen
  • HybEZ Oven (Advanced Cell Diagnostics) with slide holder and covered tray
  • Whatman 3MM chromatography paper
  • Coverslips
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps.

Alternate Protocol 1: Hybridization Histochemistry for Simultaneous Detection of Two Transcripts Using Sets of Oligodeoxynucleotide Probe Pairs

  Materials
  • 50% (v/v) formamide/4× SSPE ( appendix 2A)
  • 12‐μm cryostat tissue sections mounted on Superfrost Plus slides (Fisher) and defatted (unit 1.1)
  • Hybridization solution (see recipe)
  • 35S‐ or digoxigenin‐labeled riboprobe (see protocol 10)
  • 1× SSPE/1 mM dithiothreitol (DTT)
  • 4× SSPE/1 mM DTT
  • RNase A solution (see recipe)
  • 0.1× SSPE/1 mM DTT
  • 1× SSPE
  • 70% (v/v) ethanol
  • Sterile Bio‐Assay dishes (Nunc; 245 × 245 × 30 mm)
  • Whatman 3MM chromatography paper
  • Glass coverslips
  • 55° and 37°C incubators
  • Staining dishes with slide racks
  • 65°C water bath
  • NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps.

Basic Protocol 3: Hybridization Histochemistry with RNA Probes

  Materials
  • Digoxigenin‐labeled sections on slides (see protocol 1 or 3)
  • TBS, pH 7.5 ( appendix 2A)
  • Detection buffer (see recipe)
  • Alkaline phosphatase (AP)–conjugated sheep polyclonal anti‐digoxigenin antibody (Sigma‐Aldrich)
  • Development buffer (see recipe)
  • NBT/BCIP working solution (see recipe)
  • 1× SSPE (appendix 2a)
  • Cytoseal 60 (Stephens Scientific) or similar organic‐basic mounting medium
  • Staining dishes with slide racks
  • Slide warmer
NOTE: Carry out all steps at room temperature.

Basic Protocol 4: Detection of Digoxigenin‐Labeled Probes Using AP‐Conjugated Antibodies

  Additional materials (also see protocol 5)
  • Horseradish peroxidase (HRP)–conjugated sheep polyclonal anti‐digoxigenin antibody (Sigma‐Aldrich)
  • TSA Plus Biotin kit (Perkin‐Elmer) containing:
  • Plus Biotin TSA reagent
  • 1×Plus Amplification Diluent
  • Streptavidin‐fluorochrome (Perkin‐Elmer)
  • 0.5% bovine serum albumin (BSA)
  • Streptavidin‐HRP conjugate (Perkin‐Elmer)
  • Diaminobenzidine (DAB) tablets (Sigma)
  • Urea/hydrogen peroxide tablets (Sigma)
  • TBS, pH 8.0 ( appendix 2A)
  • Streptavidin–alkaline phosphatase (AP) conjugate (Perkin‐Elmer)
  • Cytoseal 60 (Thermo Scientific) or similar organic‐based mounting medium

Alternate Protocol 2: Detection of Digoxigenin‐Labeled Probes Using Tyramide Signal Amplification (TSA)

  Materials
  • Defatted 12‐μm fresh‐frozen or paraffin‐embedded cryostat sections (unit 1.1) or whole fixed cells, mounted on Superfrost Plus slides (Fisher)
  • 1% (w/v) paraformaldehyde/picric acid solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.1×, 0.5×, 1×, and 2× SSC ( appendix 2A)
  • Citrisolv (Fisher) or xylene
  • 70%, 80%, 95%, and 100% (v/v) ethanol
  • Citra Plus, pH 6 (BioGenex)
  • Blocking solution: PBS containing 1% (w/v) BSA and 0.6% (v/v) Triton X‐100
  • Primary antibody of interest
  • Biotinylated secondary antibody (Vectastain ABC kit, Vector Laboratories, or Jackson Immunoresearch)
  • Streptavidin‐HRP (Perkin‐Elmer)
  • 0.1 M Tris·Cl, pH 8 ( appendix 2A)
  • Biotinylated tyramide (Renaissance TSA Indirect kit; Perkin‐Elmer)
  • 0.2 N HCl
  • 70% (v/v) formamide/2× SSC
  • Hybridization solution (see recipe)
  • Digoxigenin‐labeled riboprobe (see protocol 10) to a mouse repeat sequence (Bishop and Hatat, ; Mezey et al., ) or human Y‐chromosomal marker DYZ1 (Mezey et al., )
  • Fluorochrome‐labeled streptavidin
  • Staining dishes with slide racks
  • Plastic Coplin jars
  • Sterile Bio‐Assay dishes (Nunc, 245 × 245 × 30 mm)
  • 55°, 65°, and 80°C incubators
  • 65° and 80°C water bath
  • 80°C hot plate

Basic Protocol 5: Detection of Y Chromosome DNA Using Riboprobes

  Materials
  • Ilford K5.D or Kodak NTB‐3 nuclear emulsion
  • 7.5 M ammonium acetate
  • 35S‐labeled sections on slides (see protocol 1 or 3)
  • Kodak D‐19 photographic developer, 17°C
  • Kodak Rapid Fix (without hardener), 17°C
  • Counterstain (optional): e.g., 0.4% toluidine blue, 2 μg/ml ethidium bromide, hematoxylin/eosin
  • 70% (v/v) ethanol
  • Cytoseal 60 (Stephens Scientific) or similar organic‐based mounting medium
  • Darkroom with safelight
  • Coplin jars
  • 40°C water bath
  • Clean slide
  • Black slide boxes
  • Desiccant capsules (e.g., Humi‐caps from United Desiccants–Gates)
  • Black photography tape
  • Slide racks
  • Slide warmer

Basic Protocol 6: Detection of Radiolabeled Probes

  Materials
  • 5× tailing buffer (see recipe)
  • Oligodeoxynucleotide to be used as probe
  • Labeled nucleotide (select one):
  • [α‐35S]dATP (>1000 Ci/mmol; Perkin‐Elmer)
  • mix of 250 μM digoxigenin‐11‐UTP/1 mM dNTP (or dATP; components from Roche; store mix indefinitely at –20°C)
  • Terminal deoxynucleotidyl transferase (TdT; Roche or Invitrogen)
  • TE buffer, pH 7.6 ( appendix 2A)
  • 4 M NaCl
  • 25 μg/μl yeast tRNA
  • 70% and 100% (v/v) ethanol
  • 5 M dithiothreitol (DTT)

Support Protocol 1: Preparation of Oligodeoxynucleotide Probes for Hybridization Histochemistry

  Materials
  • Linearized plasmid or PCR product containing cDNA to be transcribed
  • Labeled nucleotide (select one):
  • [α‐35S]UTP (>1000 Ci/mmol; Perkin‐Elmer)
  • 1 mM UTP/4 mM digoxigenin‐11‐UTP mix (ingredients from Roche; store mix indefinitely at –20°C)
  • 5× transcription buffer (see recipe)
  • 100 mM and 5 M dithiothreitol (DTT)
  • 10 mM individual solutions of ATP, CTP, and GTP
  • RNasin (Promega)
  • 10 to 20 U/μl RNA polymerase (SP6, T3, or T7)
  • 1 U/μl RNase‐free DNase I ( appendix 2A)
  • TE buffer, pH 7.6 ( appendix 2A)
  • 4 M NaCl
  • 25 μg/μl yeast tRNA
  • 70% and 100% (v/v) ethanol
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • TE buffer/5% (w/v) SDS
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents.

Support Protocol 2: Preparation of RNA Probes for Hybridization Histochemistry

  Materials
  • RNA sample
  • GeneScreen membrane (Perkin‐Elmer)
  • Northern hybridization solution (see recipe)
  • 35S‐labeled oligodeoxynucleotide probe (see protocol 9) or riboprobe (see protocol 10)
  • 1× SSPE ( appendix 2A)/0.2% SDS (for oligodeoxynucleotide probes)
  • 0.1× SSPE/0.2% SDS (for riboprobes)
  • UV cross‐linker (e.g., Stratalinker from Stratagene)
  • 70°C vacuum oven
  • Hybridization bags or roller bottles
  • Water bath(s) or hybridization oven(s) at 37° and 55°C (for oligodeoxynucleotide probes) or 65°C (for riboprobes)
  • Plastic wrap
  • Autoradiography cassettes
  • Intensifying screens
  • Autoradiography film (e.g., Kodak Biomax MR)
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents.
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Figures

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Literature Cited

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