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Dear George,
Did Bob say why the xylene was important?  I would  expect that BABB  
would be as alcohol-soluble as methyl salicylate:benzyl benzoate.   
Maybe the xylene was ensuring the last of the water is out, but  
thorough dehydration should be sufficient.  Many mixtures of solvents  
are given in this book: Spalteholz, W., 1914. Über das  
Durchsichtigmachen von menschlichen und tierischen Präparaten und  
seine theoretischen Bedingungen. Hirzel, Leipzig.

Glad to hear someone else has tried TDE. Do you notice any tissue  
shrinkage? How are you storing it?  I've haven't yet opened my  
bottle.  One post doc is using my mixture of MSBB on 1200 µm brain  
slices, but I'm going to try TDE on brain slices <400 µm with grad  
student. Thinner slices tend to curl after dehydration.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
[log in to unmask]

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On Feb 7, 2008, at 6:58 PM, George McNamara wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Judy,
>
> I was impressed with 2,2'-thiodiethanol, used neat (R.I. 1.52;  
> Sigma-Aldrich). Completely black background (N=2 slides, one  
> confocal session). See Staudt, Hell et al 2007 Microsc Res Tech for  
> original reference. They used RI 1.515 by adding 3% H2O. I figured  
> no reason to pollute the TDE with any additives. One oddity was  
> that by eye, both blood vessel elastin fiber autofluorescence was  
> decreased (488 nm excitation, green emission) and the DAPI nuclear  
> counterstain was quenched. Both came through nicely in the confocal.
>
> Robert Zucker has several confocal papers on BABB - benzyl alcohol/ 
> benzyl benzoate as a clearing agent (see another responder's  
> message for other names). Bob told me the key is to completely  
> dehydrate the specimen through EtOH steps into xylene, before going  
> to BABB. See his papers for exact steps.
>
> best wishes,
>
> George
>
>
> At 11:09 AM 2/5/2008, you wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> What's the best method of making a sample more transparent to  
>> reduce scattering, without changing its chemistry too much? (other  
>> than methyl salicylate and glycerol).
>>
>> Thanks.
>> Judy
>>
>> Judy Trogadis
>> Bio-Imaging Coordinator
>> St. Michael's Hospital, 7Queen
>> 30 Bond St.
>> Toronto, ON M5B 1W8, Canada
>> ph:  416-864-6060  x6337
>> pager: 416-685-9219
>> fax: 416-864-6043
>> [log in to unmask]
>>
>>
>> >>> Bill Miller <[log in to unmask]> 02/04/08 9:16 PM >>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> ultimately the lower NA with its longer working distance wins if the
>> sample is clear enough..
>>
>>
>> At 08:31 PM 2/4/2008 -0500, you wrote:
>> >Search the CONFOCAL archive at
>> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> >Hello all!
>> >
>> >I was wondering for which one penetration depth is higher: NA:1.2
>> >60x lens or NA : 0.3 10x lens?
>> >
>> >thanks
>> >Sarah
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [log in to unmask]
> [log in to unmask]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc  
> (Analytical Imaging Core Facility)