Re: How "deep" can you see in a brain slice?

Posted by Neeraj Gohad-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/How-deep-can-you-see-in-a-brain-slice-tp7256661p7259208.html

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Since we are on the topic, this question is for folks who have tried or regularly use the Sacle-A2 method of clearing tissues. In the original Hama et al. paper they have prescribed the pH of the Scale-A2 to be 7.7.  Scale-A2 is not a great buffer and it's difficult to adjust the pH to 7.7, has anybody ran into the same problem?

Best,

Neeraj.


Neeraj V. Gohad, Ph.D.
Research Assistant Professor
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: [phone]
Fax: [phone]





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Monday, February 06, 2012 11:35 AM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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Dear Dr. Busek--

What do you want to see?  The presence or absence of glia, or details on the glial cells themselves?  If all you want to do is to establish the presence of the glia, you may be able to get reasonably good results by mounting the tissue between two coverslips and taking images from either side of the "sandwich".  Gel-coat a coverslip, mount the tissue on it, and then dehydrate, clear, and mount with another coverslip using DPX.
I'd image the tissue using oil-immersion optics, bearing in mind that you want the longest working distance possible.  --Different manufacturers' objectives have different capabilities in that regard and you can't necessarily trust the specifications.

By mounting the tissue between two coverslips, you can get good optical access to either side of the tissue.  You simply place the coverslip "sandwich" on a slide and hold it in place with cellophane tape.  If you don't need to create a single image through the entire thickness of the tissue, it should work.

Good luck!

Martin Wessendorf

On 2/5/2012 1:53 PM, Petr Busek wrote:

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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 [phone]

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Martin Wessendorf, Ph.D.                   office: [phone]
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