Re: optical clearing of tissue

Posted by Russell Spear on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-optical-clearing-of-tissue-tp3783665p3788239.html

Hi

Could you use some thing like saturated chloral hydrate in water (you'll
need a drug permit), or a lactic acid/glycerol solution both are water
miscible. I use these on plant material quite often.  The major problem
is having to image in water, can you use an objective made for glycerin
immersion?

Russ

Michael Weber wrote:

> 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: [phone]   Fax: [phone]
>>> e-mail: [hidden email]
>>>
>>> Mail address: USEPA,ORD,NHEERL,TAD
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>>>
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>>>
>>>   From:       Michael Weber
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>>>                                                                                                                        
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>>>   To:        
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>>>   Date:       10/07/2009 11:56
>>> AM                                                                                      
>>>
>>>                                                                                                                        
>>>
>>>   Subject:    optical clearing of
>>> tissue                                                                              
>>>
>>>                                                                                                                        
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>>>   Sent by:    Confocal Microscopy List
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>>>
>>>
>>>
>>>
>>>
>>>
>>> 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
>>
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