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Hi,
Ok, so it is a dehydration issue. Incomplete dehydration of tissue
passing through xylene will also cause Permount and DPX to become
cloudy, a few days following coverslipping. I've been rigorous on
dehydration and passing through graded mixtures of absolution EtOH
and the MSBB mixture without cloudiness. Xylene tends to make
tissues brittle. Yeah, the BB mixtures dissolve almost anything,
even makes temporary wells of silicone grease very runny. I've been
mounting in aluminum frames used to support laser microdissection
films to which I've attached a measured 24x60 mm coverglass using
silicone aquarium glue. So far, they've survived a year or so.
I wonder if storing TDE at in aliquots at -80 would avoid the need
to fill the bottle with inert gas after opening. The class of
compounds to which TDE belongs is supposed to have some higher RI
members, which would be helpful for high RI samples.
thanks,
Glen
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Glen,
>
> Bob Zucker told me that if the tissue still has any water in it,
> the BABB will turn cloudy. Another item to be aware of with BABB is
> that it can solubilize some glues, so using it in an imaging
> chamber could results in leaks. Bob also emphasized that BABB is a
> very bad thing to have land on an objective lens or any other
> optic. One last item about BABB - I found some chemical company web
> sites that listed the refractive indices of several benzyl X
> compounds. Some are at or below RI=1.515. So, it may be feasible to
> find a mixture of two that is right at 1.515. That said, I'm
> excited about TDE because even neat, its RI is close to that of
> immersion oil and glass as to not matter except maybe hyper-
> critical deconvolution.
>
> I am currently storing the TDE (25 mL bottle, smallest that Sigma-
> Aldrich sells) in the chemical cabinet. I will probably aliquot it
> out into 0.5 or 1.0 mL eppendorf tubes to make it easier to
> distribute to users who want to try.
>
> My first user had ~5 um lung tissue sections. No way for me to tell
> whether they had shrinkage. There was an excellent article in one
> of the trade magazines (Microscopy Today?) a year or so on
> paraformaldehyde concentration in perfusion fixation. That, and
> other articles, lead me to expect that shrinkage comes from the PFA
> step, not from the later tissue clearing and dehydration.
>
> George
>
>
>
> At 11:25 AM 2/8/2008, you wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> 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]
>>
>> *********************************************************************
>> *** ******
>> The box said "Requires WindowsXP or better", so I bought a Macintosh.
>> *********************************************************************
>> *** ******
>>
>>
>> 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)
>
>
>
>
>
>
> 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)
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