Show simple item record

dc.contributor.authorCavanagh, Brentonen_US
dc.contributor.authorMeedeniya, Adrianen_US
dc.contributor.authorMuller, D.en_US
dc.contributor.authorBlumenstein, Michaelen_US
dc.contributor.authorMackay-Sim, Alanen_US
dc.date.accessioned2017-05-03T15:06:13Z
dc.date.available2017-05-03T15:06:13Z
dc.date.issued2007en_US
dc.date.modified2008-04-13T23:32:28Z
dc.identifier.urihttp://hdl.handle.net/10072/17670
dc.description.abstractKey words: neuroscience, stereology, optical sectioning, immunofluorescence. Aims: The aim was to combine unbiased cell quantification and immunofluorescence detection methods to quantify the number of tyrosine hydroxylase immunoreactive neurons in a midbrain nucleus. Background: Commonly used methods of stereological estimation [1] of cell numbers limit analyses to two cell markers per tissue section [2]. We introduce here a robust and reproducible 3D stereological method using multiple fluorescence probes that makes it possible to quantify and discriminate between multiple cell types in the same specimen. Methods: Perfusion-fixed rat brain was embedded in polyethylene glycol and serially sectioned at 35孠through the substantia nigra (SN) and ventral tegmental area (VTA) (Figure 1). A Zeiss Z1 microscope combined with an ApoTome was used for optical sectioning, and Zeiss Axiovision software for image analysis. Specimens were processed for single or multiple labelling immunofluorescence and all cell nuclei were counterstained with a fluorescent DNA marker. Sections were imaged and analysed using a systematic random-sampling strategy for unbiased stereological quantification of cell numbers [3], modified for 3D reconstructed images. Results: Dopaminergic neurons in SN and VTA were identified with an antibody to the enzyme tyrosine hydroxylase and the nuclear stain DAPI. Using a semi-automated 3D sampling procedure, a total of 24,614 dopaminergic neurons was calculated in this region, a number consistent with previous methods using standard stereological methods. Double immunofluorescence labelling with the calcium binding protein, calbindin, allows analysis of neuronal subpopulations in this brain region. Summary & Conclusions: Polyethylene glycol embedding combined with immunofluorescence and automated 3D wide-field microscopy greatly facilitates discrimination of cell types within the brain. When combined with automated sampling and 3D reconstruction this greatly enhances the power of stereological analysis to quantify multiple cell types in the same tissue sections. Reference: 1. Geuna, S., Appreciating the difference between design-based and model-based sampling strategies in quantitative morphology of the nervous system. J Comp Neurol, 2000. 427(3): p. 333-9. 2. Oorschot, D.E., Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal ganglia: a stereological study using the cavalieri and optical disector methods. J Comp Neurol, 1996. 366(4): p. 580-99. 3. Howard, V., M.G. Reed, and Royal Microscopical Society (Great Britain), Unbiased stereology : three-dimensional measurement in microscopy. Microscopy handbooks ; 41. 1998, Oxford: Bios Scientific in association with the Royal Microscopical Society. xvii, 246.en_US
dc.description.publicationstatusYesen_AU
dc.languageEnglishen_US
dc.language.isoen_AU
dc.publisherNo data provideden_US
dc.relation.ispartofstudentpublicationYen_AU
dc.relation.ispartofconferencenameFocus on Microscopy Congressen_US
dc.relation.ispartofconferencetitleIntroducing “Fluorescence Neurosteriology”: Novel methods for mapping the brain.en_US
dc.relation.ispartofdatefrom2007-04-10en_US
dc.relation.ispartofdateto2007-04-13en_US
dc.relation.ispartoflocationValencia, Spainen_US
dc.rights.retentionYen_AU
dc.subject.fieldofresearchcode320799en_US
dc.titleIntroducing “Fluorescence Neurosteriology”: Novel methods for mapping the brain.en_US
dc.typeConference outputen_US
dc.type.descriptionE3 - Conference Publications (Extract Paper)en_US
dc.type.codeE - Conference Publicationsen_US
gro.date.issued2007
gro.hasfulltextNo Full Text


Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item appears in the following Collection(s)

  • Conference outputs
    Contains papers delivered by Griffith authors at national and international conferences.

Show simple item record