http://confocal-microscopy-list.275.s1.nabble.com/How-deep-can-you-see-in-a-brain-slice-tp7256661p7257463.html
I've seen. You would want to make sure to have a long-working distance
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
>
> Fixed and cleared: all the way:
>
> Three-dimensional imaging of the unsectioned adult spinal cord to assess
> axon regeneration and glial responses after injury. </pubmed/22198277>
> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N,
> Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F.
> Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277
>
> Scale: a chemical approach for fluorescence imaging and reconstruction of
> transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H,
> Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat
> Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>
> A colleague here at the U told me his lab had much better clearing and
> imaging with the Erturk et al method than with the versions of Hama et al's
> Scale that they tried (no, I do not know which many variants they tried or
> how extensively they tested each). This colleague told me that with the
> Erturk et al method they needed to image the same day (and the sooner the
> better). The Erturk et al method uses tetrahydrofuran (THF) to strip the
> lipids from the tissue, followed by immersion in benzyl alcohol:benzyl
> benzoate (BABB). BABB has a long history of use in optical clearing - see
> various papers by Bob Zucker, for examples:
>
> Whole insect and mammalian embryo imaging with confocal microscopy:
> morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006
> Nov 1;69(11):1143-52. PMID: 17051584
>
> Confocal laser scanning microscopy of whole mouse ovaries: excellent
> morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>*
> *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804
>
> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better
> ultimate destination after THF. For TDE see:
>
> 2,2'-thiodiethanol: a new water soluble mounting medium for high
> resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC,
> Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID:
> 17131355
>
> See also Stan Vitha's post(s) here on transitioning specimens into TDE and
> imaging.
>
>
> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse,
> flush the RBCs, take out the brain, slice in half (along a line that will
> bisect the glioma mass that you introduced by stereotaxic injection, being
> careful not to have cells up the needle track), bring to the confocal - a
> user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably
> 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image
> hemisectioned mouse brains previously implanted with GFP+ neural stem cells
> (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm
> excitation) several hundred micrometers deep. Again, one of the keys is to
> flush out the blood cells from the mouse vasculature - they scatter a lot
> more than mouse brain tissue. I have never been involved with brain slices
> - hopefully those protocols flush the blood cells after sac'ing the mouse.
>
> George
>
>
>
>
>
> On 2/5/2012 2:53 PM, Petr Busek wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
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>> *****
>>
>> Dear all,
>> I am trying to view fluorescently labeled glioma cells invading into a
>> 400um
>> thick brain slice on an Olypus FV300. Has anyone experience with this and
>> how "deep" it is reasonable to expect to see in the slice using a confocal
>> microscope? How can you maximize this depth? (selection of objectives,
>> processing of the slice....)
>> Thanks for any suggestions, Petr.
>>
>> Petr Busek, MD, PhD
>> Charles University in Prague
>> First Faculty of Medicine
>> Laboratory of Cancer Cell Biology
>> Institute of Biochemistry and Experimental Oncology
>> U Nemocnice 5
>> 128 53 Prague 2
>> Czech Republic
>> www.lf1.cuni.cz/lbnb
>> Fax +420 224 965 826
>>
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami
>