Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ Hybridization (catFISH)

John F. Guzowski1, Paul F. Worley2

1 University of Arizona, Tucson, Arizona, 2 Johns Hopkins University, School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.8
DOI:  10.1002/0471142301.ns0108s15
Online Posting Date:  August, 2001
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Abstract

This protocol describes a method (cellular compartment analysis of temporal activity by fluorescent in situ hybridization or catFISH) that uses fluorescent in situ hybridization to immediate‐early gene RNAs and confocal microscopy to identify neuronal populations activated at two distinct times. The combination of cellular and temporal resolution makes catFISH a valuable tool for investigating the dynamic interactions of neuronal populations associated with different behaviors or cognitive challenges.

     
 
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Table of Contents

  • Basic Protocol 1: Dectection of Immediate Early Genes by Fluorescence In Situ Hybridization and Nuclear Counterstaining
  • Basic Protocol 2: Acquisition of Confocal Image Series
  • Basic Protocol 3: Cellular Analysis of Confocal Image Series
  • Alternate Protocol 1: Quantification of Total Nuclear and Extranuclear Immediate Early Gene RNA Signal
  • Support Protocol 1: Preparation of Slide‐Mounted Tissue Sections for catFISH
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Dectection of Immediate Early Genes by Fluorescence In Situ Hybridization and Nuclear Counterstaining

  Materials
  • Commercial in vitro transcription kit (e.g., MAXIscript; Ambion)
  • Hapten‐modified RNA labeling mix (e.g., DIG RNA or fluorescein labeling mix; Roche Diagnostics)
  • Plasmid template, linearized with the appropriate restriction enzyme ( appendix 1M)
  • RNA gel loading buffer (provided in MAXIscript kit; optional): 95% (v/v) formamide/0.025% (w/v) xylene cyanol/0.025% (w/v) bromphenol blue/18 mM EDTA/0.025% (w/v) SDS
  • RNA molecular weight marker
  • Slides with 20‐µm tissue sections (see protocol 5), stored in box at −70°C
  • 4% (w/v) buffered paraformaldehyde solution (see recipe), ice cold
  • 20× SSC solution (dilute as needed with nuclease‐free water; Sigma or appendix 2A)
  • Acetic anhydride solution (see recipe)
  • DEPC‐treated H 2O ( appendix 2A)
  • 1:1 (v/v) acetone/methanol solution, ice cold
  • 1× prehybridization solution (Sigma): dilute 2× concentrate 1:1 (v/v) with deionized formamide
  • 2× SSC/50% (v/v) deionized formamide
  • 1× hybridization solution (Amersham Pharmacia Biotech), supplied as a 2× concentrate; dilute 1:1 (v/v) with deionized formamide
  • 10 µg/ml RNase A in 2× SSC
  • 2% (v/v) H 2O 2/1× SSC (optional)
  • Tris‐buffered saline (TBS; appendix 2A)
  • TSA‐Direct System (NEN Life Sciences)
  • Fluorescent nuclear counterstain (e.g., DAPI, propidium iodide, YOYO‐1; Molecular Probes)
  • Aqueous mounting medium with anti‐fade agent (e.g., Vectashield, Vector Laboratories)
  • Clear nail polish
  • Micro‐spin columns (e.g., mini Quick Spin RNA columns; Roche Diagnostics)
  • Heating block, 90°C
  • Slide staining dishes and racks
  • Whatman 3MM filter paper
  • Glass coverslips (e.g., Corning)
  • Chamber for slide incubation
  • Hybridization oven, 56°C
  • 37° and 56°C water baths
  • Additional reagents and equipment for denaturing agarose (CPMB UNIT ) or polyacrylamide gel electrophoresis (CPMB UNIT ) or agarose minigel electrophoresis (CPMB UNIT )
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps. Use deionized water in subsequent probe detection steps.

Basic Protocol 2: Acquisition of Confocal Image Series

  Materials
  • Slides with catFISH‐processed tissue sections (see protocol 1)
  • Confocal microscope with two photomultiplier tubes (PMTs) and computer interface
  • Computer and computer storage medium

Basic Protocol 3: Cellular Analysis of Confocal Image Series

  Materials
  • Confocal image series file, saved using RGB format (see protocol 2)
  • Computer with image analysis software that can create image stacks (e.g., MetaMorph, Universal Imaging; NIH Image, National Institutes of Health; Scion Image for Windows, Scion; The Zeiss Image Browser [Carl Zeiss]. NIH Image, Scion Image for Windows, and The Zeiss Image Browser are available at no cost)
  • Overhead transparencies and markers

Alternate Protocol 1: Quantification of Total Nuclear and Extranuclear Immediate Early Gene RNA Signal

  Materials
  • Confocal image series file, saved using RGB format (see protocol 2)
  • Computer with image analysis software that can perform threshold and calculation functions on separate color channels in an RGB image (e.g., Adobe Photoshop, Adobe Systems; MetaMorph, Universal Imaging)

Support Protocol 1: Preparation of Slide‐Mounted Tissue Sections for catFISH

  Materials
  • Experimental animals
  • Isopentane (2‐methylbutane; Aldrich)
  • Dry ice/ethanol slurry
  • Freezing medium (e.g., OCT; VWR Scientific Products)
  • Dissecting tools (unit 1.1)
  • Sealable storage bags
  • Brain matrix (Ted Pella), optional
  • Cryostat and accessories, including molds, disposable cryostat blades, and chucks, −20°C
  • Microscope slides (Superfrost Plus, VWR Scientific Products)
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Figures

Videos

Literature Cited

Literature Cited
   Cole, A.J., Saffen, D.W., Baraban, J.M., and Worley, P.F. 1989. Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340:474‐476.
   Greenberg, M.E., Ziff, E.B., and Greene, L.A. 1986. Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 234:80‐83.
   Guzowski, J.F., McNaughton, B.L., Barnes, C.A., and Worley, P.F. 1999. Environment‐specific expression of the immediate‐early gene Arc in hippocampal neuronal ensembles. Nature Neurosci. 2:1120‐1124.
   Guzowski, J.F., Lyford, G.L., Stevenson, G.D., Houston, F.P., McGaugh, J.L., Worley, P.F., and Barnes, C.A. 2000. Inhibition of activity‐dependent Arc protein expression in the rat hippocampus impairs the maintenance of long‐term potentiation and consolidation of long‐term memory. J. Neurosci. 20:3993‐4001.
   Link, W., Konietsko, U., Kauselmann, G., Krug, M., Schwanke, B., Frey, U., and Kuhl, D. 1995. Somatodendritic expression of an immediate‐early gene is regulated by synaptic activity. Proc. Natl. Acad. Sci. U.S.A. 92:5734‐5738.
   Lyford, G.L., Yamagata, K., Kaufmann, W.E., Barnes, C.A., Sanders, L.K., Copeland, N.G., and Worley, P.F. 1995. Arc, a growth factor and activity‐regulated gene, encodes a novel cytoskeleton‐associated protein that is enriched in neuronal dendrites. Neuron 14:433‐445.
   Morgan, J.I., Cohen, D.R., Hempstead, J.L., and Curran, T. 1987. Mapping patterns of c‐fos expression in the central nervous system after seizure. Science 237:192‐197.
   Steward, O., Wallace, C.S., Lyford, G.L., and Worley, P.F. 1998. Synaptic activation causes the mRNA for the immediate early gene Arc to localize selectively near activated postsynaptic sites on neuronal dendrites. Neuron 21:741‐751.
   Wallace, C.S., Lyford, G.L., Worley, P.F., and Steward, O. 1998. Differential intracellular sorting of immediate‐early gene mRNAs depends on signals in the mRNA sequence. J. Neurosci. 18:26‐35.
   Worley, P.F., Bhat, R.V., Baraban, J.M., Erickson, C.A., McNaughton, B.L., and Barnes, C.A. 1993. Thresholds for synaptic activation of transcription factors in hippocampus: Correlation with long‐term enhancement. J. Neurosci. 13:4776‐4786.
Key Reference
   Guzowski et al., 1999. See above.
  Describes the basic biological findings that enable the catFISH approach and uses two‐epoch catFISH to demonstrate that Arc gene expression is specifically induced by neural encoding processes in hippocampal CA1 neurons.
Internet Resources
   http://www.zeiss.com/micro/products/
  The Zeiss LSM Image Browser software program can be downloaded for free from this site. The Image Browser is simple to use and works very well for analyzing image stacks. There are also links to other resources on microscopy.
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