Leica and Abberior say: ¨Please do not use Vectashield, Vectashield Hard set or (other) embedding media containing p-phenylenediamine as antifading reagent." (I do not understand why, but I have no choice to trust them)
Prolong Gold transforms the cells (10 - 12 um of the thickness) in 2 um pancake
The list of allies of a microscopist becomes short ...
I also do not recommend Vectashield for super resolution. I have seen strange artifcacts in the z dimension. It almost looks like some sort of stratification of refractive index causing distortion. Prolong works in my experience. Be gentle when you mount the coverslip.
In the end, you want to match to the refractive index of the immersion oil. Stefan Hell's group published a paper about using TDE as a mounting medium and you can exactly get the refractive index matched to 1.5. Abberior also cells several types of liquid and hardening mounting medium. One they recommend for 3D STED, I think the hardening one.
Hi. I have used Prolong Gold when imaging samples using STED and it has worked very well. There is minimal sample distortion and it is readily available. Make sure to follow the manufacturer's advice with regards to the curating period (>24h) and to use high quality coverslips.
I would recommend to use 90% glycerol supplemented with 5% (w/v) NPG (N-propyl gallate). This medium is not hardening, so you would need to use either spacers (it also helps to prevent shrinkage of your samples) or nail polish to seal the edges of the cover slip.
Thank you, all
I'm wondering about NPG. Is it really protective against depletion lasers? And by the way, how do you dissolve it? Do you heat or sonicate?
thanks
It appeared to be better than DABCO or Fluoromount G hardening medium in terms of bleaching. We were heating and mixing for long period. If the preparation was correct and NPG was new, medium will be translucent (once I got yellow because of expired NPG).
Dear Leonid,
I recently attended a seminar from Thermo Fisher on Molecular Probes dyes and products for fluorescence microscopy. Apparently, vectashield quenches certain dyes while it is good for others and it comes with a certain auto-fluorescence . Prolong Gold is a mixture produced to work for a broad spectrum of dyes while Prolong Diamond should also protect fluorescent proteins. Recently, a new Prolong version called Glass came out with a refractive index of 1.52. Gold and Glass are curing media while Diamond is glycerol-based and stays liquid so you have to seal it.
I used Prolong Diamond for fixed cells in STED and was happy with it but when my PhD student used it to mount filter support with polarized cells he also faced some cell squeezing depending how he mounted the filter.
If you can, I would suggest use Prolong Diamond and a spacer if necessary.
Cheers and good luck
Thank you, Claus, I'm going to read on the Diamond and then I'll try it.
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