Advice on mounting media:

The following is for coverslips mounted on slides and is better written than enything I could provide.

I would add, however, measuring the amount, such as trying 10 ul for a 18x18 mm coverlsip.

I don't know what the favorite home brew in email below is, but this is what I was taught back in the day: (final volume 5 ml) N-Propyl gallate 6 g/l in glycerol 50% in TBS or PBS 1X pH 8.0. Add the N-propyl gallate to 2.5 ml of glycerol the day before and let it dissolve overnight at RT (or use a lot of vortexing). Add 2.5 ml of 2X TBS/PBS on the day of the experiment. I never did this, but have been told, 0.02% NaN₃ can also be added to the final mix for better preservation.

But I've found for most imaging with the new bright dyes, just plain PBS works fine.

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Hi David and Fred,

I too am a fan of Prolong Diamond.  It works better than Prolong Gold on blue-emitting dyes and also on the fluorescent proteins. We do not allow it to cure, otherwise it squishes everything flat (as does Mowiol) which is obviously no good for 3D-imaging.  But if you just wick away the excess for a minute and then seal immediately around the edge of the coverslip, it works really well on all of our microscopes.

For some other dyes (non-Alexa), like the Bodipy dyes and also some blue dyes like DyLight 405, I find that home-made mountants containing N-propyl gallate work best.  It all depends on the exact dye - for example, Vectashield kills Alexa 647, while DABCO kills Alexa 488 - they knock the initial intensities down by about 75%, according to a graph I got from Molecular Probes, and we've certainly seen that in practice for Vectashield and AF647.  I believe that Vectashield contains Phenylene Diamene (though they won't tell you), which is great for FITC but terrible for far red dyes, nor so good for some of the red Alexa dyes e.g. 594.

Best,

Alison

On 3/28/2018 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] wrote:
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> 
> Dear David,
> 
> 
> Try DABCO for home-made, I can provide a recipe if you wish.
> 
> 
> Among commercial anti-fade mounting media, we found that Prolong
> Gold/Diamond works best for our applications
> in both confocal and iSIM (not so different from your spinning disc).
> 
> 
> Best Wishes,
> 
> Fred
> 
>