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hey stanislav,
it is not mandatory to use tde with the highest purity grade. Sometimes, the lower grade tde is a bit more acidic, but that does not matter, since one has to adjust the ph anyway (if you are lucky, you get a batch with ideal ph). The lower grade TDE is sometimes yellowish coloured, so it might contain uv or blue light absorbing impurities, fluorescing green. But in general, the concentration of absorbing or fluorescent entities in tde is extremly low. Because of that, tde is sometimes used as an immersion oil. There is also no evidence of enhanced photobleaching using lower grade tde. So if it works fine, i would not change the tde to higher grade tde. One reason to use the highest grade tde is that you might have more reliable conditions.
As antifades, you can add a small amount of DABCO (1mM). The best way to do that is to solve dabco in the water you use to adjust the refractive index of tde to 1.517.
By the way, there is a new protocol for adjustin the ph:
use liquid pH indicators like Phenol red or bromthymolblue.
(phenolred changes from yellow to redviolet at pH 7.3; bromthymolblue changes from green to blue at 7.5)
Make a reference set of different pH values (6.5 to 8 in 0.2 steps)(water, ph adjusted
with naoh or hcl, measured with a ph electrode) and the indicators and compare the colors
with 10ml tde plus indicator. Then, add small amounts of base or acid until you have reached
the color (by comparing with the reference set) corresponding to the desired pH. Because you are examining a large volume
(and not only a glas surface), the method is fast and reliable.
if you have any problem with the tde, don´t hesitate to contact me.
best wishes,
thorsten
Thorsten,
could you comment on the importance of TDE mountant purity?
Your article (MICROSCOPY RESEARCH AND TECHNIQUE 70:1-9, 2007) used the
Biochemica grade TDE (>99%, Sigma #88559, 250ML for $160.50). I have
been using the Aldrich cat. #166782 (>99%, 500g $27.70) with good success
for conofcal 3D imaging of pollen grains (autofluorescent, no staining needed).
It performed really well for these samples, but I am uncertain if this lower
grade TDE would work for e.g., immunostained slides.
Also, if anyone is using TDE with an antifade compound, I would appreciate
information what you used, what concentration, and any specific tips on
mixing.
Thank you!
Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257
On Wed, 7 Oct 2009 18:10:56 +0200, Staudt Thorsten <T.Staudt@DKFZ-
HEIDELBERG.DE> wrote:
>hello michael,
>
>This publication might be interesting for you:
>
>
>
http://www.ncbi.nlm.nih.gov/pubmed/19397748>
>
>thorsten
>
>
>-----Ursprüngliche Nachricht-----
>Von: Confocal Microscopy List im Auftrag von Michael Weber
>Gesendet: Mi 07.10.2009 17:56
>An:
[hidden email]
>Betreff: optical clearing of tissue
>
>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