title: A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.
abstract: Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.
authors: Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan.
authors: Yuka
authors: Y
authors: Aoyagi
authors: Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
authors: Ryosuke
authors: R
authors: Kawakami
authors: Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan.
authors: Hisayuki
authors: H
authors: Osanai
authors: Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
authors: Terumasa
authors: T
authors: Hibi
authors: Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
authors: Tomomi
authors: T
authors: Nemoto
mesh_terms: D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D003712:Dendrites / Q000648:ultrastructure*; D049229:Dendritic Spines / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D009474:Neurons / Q000648:ultrastructure*; D013438:Sulfhydryl Compounds
chemical_list: D013438:Sulfhydryl Compounds; C047519:2,2'-thiodiethanol
references: Sci Rep. 2013;3:1014
references: 23350026
references: Nat Protoc. 2012 Nov;7(11):1983-95
references: 23060243
references: Nat Neurosci. 2011 Aug 30;14(11):1481-8
references: 21878933
references: Nat Rev Neurosci. 2012 Jun 20;13(7):478-90
references: 22714019
references: PLoS One. 2011;6(10):e24695
references: 21984894
references: Front Neurosci. 2007 Oct 15;1(1):131-43
references: 18982124
references: Nat Photonics. 2013 Mar 1;7(3):
references: 24353743
references: Phys Med Biol. 2007 Apr 21;52(8):2035-54
references: 17404454
references: Cell. 2014 Aug 14;158(4):945-958
references: 25088144
references: Neuron. 2000 Oct;28(1):41-51
references: 11086982
references: Int J Toxicol. 2005 Nov-Dec;24(6):435-42
references: 16393936
references: Nat Med. 2011 Dec 25;18(1):166-71
references: 22198277
references: Microsc Res Tech. 2007 Jan;70(1):1-9
references: 17131355
references: PLoS One. 2014 Apr 08;9(4):e94054
references: 24714405
references: Science. 2010 Nov 26;330(6008):1198-201
references: 21109663
references: Nat Neurosci. 2013 Aug;16(8):1154-61
references: 23792946
references: Nature. 2013 May 16;497(7449):332-7
references: 23575631
references: Methods Biochem Anal. 2006;47:39-65
references: 16335709
references: Development. 2013 Mar;140(6):1364-8
references: 23444362
references: Nat Neurosci. 2011 Mar;14(3):285-93
references: 21346746
references: Nature. 2004 Jun 17;429(6993):761-6
references: 15190253
references: J Vis Exp. 2012 Sep 23;(67):e3848
references: 23023035
references: PLoS Comput Biol. 2005 Sep;1(4):e42
references: 16201007
references: Cell. 2014 Apr 24;157(3):726-39
references: 24746791
references: Opt Express. 2009 Aug 3;17(16):13354-64
references: 19654740
references: J Microsc. 2009 May;234(2):196-204
references: 19397748
references: Annu Rev Biochem. 1998;67:509-44
references: 9759496