A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections

Sathyanesan Samuel Newton, Antonia Dow, Rose Terwilliger, Ronald Duman

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion of the brain with paraformaldehyde, this protocol can be carried out on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the technique as the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB.

Original languageEnglish (US)
Pages (from-to)214-219
Number of pages6
JournalBrain Research Protocols
Volume9
Issue number3
DOIs
StatePublished - Jul 22 2002

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In Situ Hybridization
Immunohistochemistry
Cyclic AMP Response Element-Binding Protein
Brain
Paraffin Embedding
Gene Expression
Dynorphins
Enkephalins
Frozen Sections
Post Translational Protein Processing
Microarray Analysis
Transgenic Mice
Glass
Real-Time Polymerase Chain Reaction
Perfusion
Phosphorylation
Antigens
Messenger RNA

Keywords

  • Colocalization
  • Combined immunohistochemistry and in situ hybridization
  • Gene expression

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections. / Newton, Sathyanesan Samuel; Dow, Antonia; Terwilliger, Rose; Duman, Ronald.

In: Brain Research Protocols, Vol. 9, No. 3, 22.07.2002, p. 214-219.

Research output: Contribution to journalArticle

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