An optimized protocol for high-throughput in situ hybridization of zebra finch brain

Julia B. Carleton, Peter V. Lovell, Anne McHugh, Tessa Marzulla, Katy L. Horback, Claudio Mello

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-μm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

Original languageEnglish (US)
Pages (from-to)1249-1258
Number of pages10
JournalCold Spring Harbor Protocols
Volume2014
Issue number12
DOIs
StatePublished - Dec 1 2014

Fingerprint

Finches
Digoxigenin
Equidae
Gene expression
In Situ Hybridization
Brain
Throughput
Chromogenics
Tissue
Imaging techniques
Optical resolving power
Emulsions
Complementary DNA
Genes
Messenger RNA
Songbirds
Gene Expression Profiling
Tissue Distribution
Autoradiography
Costs

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

An optimized protocol for high-throughput in situ hybridization of zebra finch brain. / Carleton, Julia B.; Lovell, Peter V.; McHugh, Anne; Marzulla, Tessa; Horback, Katy L.; Mello, Claudio.

In: Cold Spring Harbor Protocols, Vol. 2014, No. 12, 01.12.2014, p. 1249-1258.

Research output: Contribution to journalArticle

Carleton, JB, Lovell, PV, McHugh, A, Marzulla, T, Horback, KL & Mello, C 2014, 'An optimized protocol for high-throughput in situ hybridization of zebra finch brain', Cold Spring Harbor Protocols, vol. 2014, no. 12, pp. 1249-1258. https://doi.org/10.1101/pdb.prot084582
Carleton, Julia B. ; Lovell, Peter V. ; McHugh, Anne ; Marzulla, Tessa ; Horback, Katy L. ; Mello, Claudio. / An optimized protocol for high-throughput in situ hybridization of zebra finch brain. In: Cold Spring Harbor Protocols. 2014 ; Vol. 2014, No. 12. pp. 1249-1258.
@article{a22fe8ef89934424845f7103fca890db,
title = "An optimized protocol for high-throughput in situ hybridization of zebra finch brain",
abstract = "In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-μm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.",
author = "Carleton, {Julia B.} and Lovell, {Peter V.} and Anne McHugh and Tessa Marzulla and Horback, {Katy L.} and Claudio Mello",
year = "2014",
month = "12",
day = "1",
doi = "10.1101/pdb.prot084582",
language = "English (US)",
volume = "2014",
pages = "1249--1258",
journal = "Cold Spring Harbor Protocols",
issn = "1559-6095",
publisher = "Cold Spring Harbor Laboratory Press",
number = "12",

}

TY - JOUR

T1 - An optimized protocol for high-throughput in situ hybridization of zebra finch brain

AU - Carleton, Julia B.

AU - Lovell, Peter V.

AU - McHugh, Anne

AU - Marzulla, Tessa

AU - Horback, Katy L.

AU - Mello, Claudio

PY - 2014/12/1

Y1 - 2014/12/1

N2 - In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-μm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

AB - In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-μm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

UR - http://www.scopus.com/inward/record.url?scp=84923831786&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84923831786&partnerID=8YFLogxK

U2 - 10.1101/pdb.prot084582

DO - 10.1101/pdb.prot084582

M3 - Article

C2 - 25342071

AN - SCOPUS:84923831786

VL - 2014

SP - 1249

EP - 1258

JO - Cold Spring Harbor Protocols

JF - Cold Spring Harbor Protocols

SN - 1559-6095

IS - 12

ER -