Dear all,
first of all, thanks for the replies off- and online!
I should have mentioned a bit more details in my initial post. The
embryos get mounted in agarose and investigated with dipping objectives
with water as medium, so the RI of the surrounding medium is around
1.33. I expect the RI of the embryo to be higher, so the limiting factor
for penetration depth is diffraction between medium and embryo. But,
there is nothing I can do about that, right? Clearing with i.e. BABB
would not make any sense, if I put the samples back in water-like medium
afterwards and do not use oil objectives anyway. So the imaging
conditions are already as optimized as possible - is that a valid
conclusion, or am I missing something?
cheers,
Michael
Phil Hertzler schrieb:
> Mike,
>
> Methyl salicylate (oil of wintergreen) also works well and smells better
> than BABB. :-) Transition through 100% ethanol from aqueous buffer.
> I've stored samples over 15 years in MS without loss of fluorescence.
>
> Best regards,
>
> Phil
>
> At 01:21 PM 10/7/2009, you wrote:
>> Mike
>> This is a review that describes our procedure of clearing mammalian and
>> insect tissue with BABB. Reprints are available on request
>>
>> Zucker, R.M.Technical note: Whole insects and Mammalian Embryo Imaging
>> with Confocal Microscopy: Morphology and Apoptosis. Cytometry 2006 69A:
>> 1143-1152
>>
>> Best wishes
>> bob
>>
>> Robert M. Zucker, PhD
>> U.S. Environmental Protection Agency
>> Office of Research and Development
>> National Health and Environmental Effects Research Laboratory.
>> Toxicology Assessment Division
>> Telephone: 919-541-1585 Fax: 919-541-4017
>> e-mail: [log in to unmask]
>>
>> Mail address: USEPA,ORD,NHEERL,TAD
>> Developmental Biology Branch ( MD 67)
>> Research Triangle Park, North Carolina, 27711
>>
>> Shipping address:
>> 2525 E.NC Highway 54
>> Durham, NC, 27713
>>
>>
>>
>>
>>
>> From: Michael Weber
>> <[log in to unmask]>
>>
>>
>>
>> To:
>> [log in to unmask]
>>
>>
>>
>> Date: 10/07/2009 11:56
>> AM
>>
>>
>>
>> Subject: optical clearing of
>> tissue
>>
>>
>>
>> Sent by: Confocal Microscopy List
>> <[log in to unmask]>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dear list,
>>
>> I am looking for advice on optical "clearing" of fixed tissue before
>> staining it and using it for light microscopy. Actually "tissue" is not
>> the precise term, since I would like to clear whole fly embryos. This
>> process seems to be well established in histology, i.e. using Xylene. I
>> also found a commercial product called "Histo-Clear" (National
>> Diagnostics), which claims to preserve tissue structures rather well,
>> while being less nasty compared to Xylene. Did you guys ever use
>> something
>> like that? Any input welcome.
>>
>> cheers,
>> Michael
>
> ------------------------------------------------------------------------
> Philip L. Hertzler
> Professor
> Central Michigan University
> Dept. of Biology, Brooks Hall 217
> 200 Library Dr.
> Mount Pleasant, MI 48859
>
> Phone: (989) 774-2393
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