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A clearer view of the insect brain - combining bleaching with standard whole-mount immunocytochemistry allows confocal imaging of pigment-covered brain areas for 3D reconstruction

Stöckl, Anna LU and Heinze, Stanley LU (2015) In Frontiers in Neuroanatomy 9.
Abstract
In the study of insect neuroanatomy, three-dimensional (3D) reconstructions of neurons and neuropils have become a standard technique. As images have to be obtained from whole-mount brain preparations, pigmentation on the brain surface poses a serious challenge to imaging. In insects, this is a major problematic in the first visual neuropil of the optic lobe, the lamina, which is obstructed by the pigment of the retina as well as by the pigmented fenestration layer. This has prevented inclusion of this major processing center of the insect visual system into most neuroanatomical brain atlases and hinders imaging of neurons within the lamina by confocal microscopy. It has recently been shown that hydrogen peroxide bleaching is compatible... (More)
In the study of insect neuroanatomy, three-dimensional (3D) reconstructions of neurons and neuropils have become a standard technique. As images have to be obtained from whole-mount brain preparations, pigmentation on the brain surface poses a serious challenge to imaging. In insects, this is a major problematic in the first visual neuropil of the optic lobe, the lamina, which is obstructed by the pigment of the retina as well as by the pigmented fenestration layer. This has prevented inclusion of this major processing center of the insect visual system into most neuroanatomical brain atlases and hinders imaging of neurons within the lamina by confocal microscopy. It has recently been shown that hydrogen peroxide bleaching is compatible with immunohistochemical labeling in insect brains, and we therefore developed a simple technique for removal of pigments on the surface of insect brains by chemical bleaching. We show that our technique enables imaging of the pigment obstructed regions of insect brains when combined with standard protocols for both anti-synapsin-labeled as well as neurobiotin-injected samples. This method can be combined with different fixation procedures, as well as different fluorophore excitation wavelengths without negative effects on staining quality. It can therefore serve as an effective addition to most standard histology protocols used in insect neuroanatomy. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
whole-mount labeling, confocal imaging, lamina, pigment, bleaching
in
Frontiers in Neuroanatomy
volume
9
publisher
Frontiers
external identifiers
  • wos:000361543500001
  • pmid:26441552
  • scopus:84941097850
ISSN
1662-5129
DOI
10.3389/fnana.2015.00121
language
English
LU publication?
yes
id
02088f9e-6b6f-4169-af05-e5816fa44d33 (old id 8071204)
date added to LUP
2015-10-22 13:13:47
date last changed
2017-04-23 04:01:02
@article{02088f9e-6b6f-4169-af05-e5816fa44d33,
  abstract     = {In the study of insect neuroanatomy, three-dimensional (3D) reconstructions of neurons and neuropils have become a standard technique. As images have to be obtained from whole-mount brain preparations, pigmentation on the brain surface poses a serious challenge to imaging. In insects, this is a major problematic in the first visual neuropil of the optic lobe, the lamina, which is obstructed by the pigment of the retina as well as by the pigmented fenestration layer. This has prevented inclusion of this major processing center of the insect visual system into most neuroanatomical brain atlases and hinders imaging of neurons within the lamina by confocal microscopy. It has recently been shown that hydrogen peroxide bleaching is compatible with immunohistochemical labeling in insect brains, and we therefore developed a simple technique for removal of pigments on the surface of insect brains by chemical bleaching. We show that our technique enables imaging of the pigment obstructed regions of insect brains when combined with standard protocols for both anti-synapsin-labeled as well as neurobiotin-injected samples. This method can be combined with different fixation procedures, as well as different fluorophore excitation wavelengths without negative effects on staining quality. It can therefore serve as an effective addition to most standard histology protocols used in insect neuroanatomy.},
  articleno    = {121},
  author       = {Stöckl, Anna and Heinze, Stanley},
  issn         = {1662-5129},
  keyword      = {whole-mount labeling,confocal imaging,lamina,pigment,bleaching},
  language     = {eng},
  publisher    = {Frontiers},
  series       = {Frontiers in Neuroanatomy},
  title        = {A clearer view of the insect brain - combining bleaching with standard whole-mount immunocytochemistry allows confocal imaging of pigment-covered brain areas for 3D reconstruction},
  url          = {http://dx.doi.org/10.3389/fnana.2015.00121},
  volume       = {9},
  year         = {2015},
}