Re: [Histonet] inconsistent DAPI staining

From:Gayle Callis

A couple of things here:

When using Prolong Gold with DAPI, you must blot away most of the PBS 
before you add the drop (generally right on top of the section) and then 
the coverslip. Failure to stain may be a dilution factor with too much 
PBS.  Do NOT seal with fingernail polish especially if you ever work with 
GFP. These were instructions directly from Tech services at Molecular Probes.

To seal a coverslip for immunofluorescent staining,  we discard fingernail 
polish out of the tidy little bottle, rinse bottle and brush with acetone, 
than let it completely dry and then add permanent mounting media diluted 
with either toluene or xylene.  We love the little brush but hate the 
fingernail polish.  We seal coverslips with diluted permanent mounting 
media that is NOT miscible with water. The reason is that fingernail polish 
contains isopropyl alcohol that leaches into the aqueous PBS under coverslip.

Be careful how you store Prolong Gold with DAPI ready to use, it must be 
stored with dessicant AND frozen, then brought to room temperature just 
before use.  We used an applicator stick to put a drop on from bottle to 
section, and try NOT to introduce any moisture back the the Prolong Gold 
stock.  We refreeze Prolong Gold several times until it is gone, and it 
works perfectly.

DAPI will stain  DNA in either fixed or unfixed frozen sections very 
nicely, but I am not sure how well it stains if the tissue has been fixed a 
long time in formalin or paraformaldehyde - we avoid that.   You did not 
say whether your tissues were fixed or fresh?  Your failure to stain with 
DAPI may not be a problem with mounting media, but with some other thing 
you are doing to the tissue, frozen section or during staining that affects 
the DAPI.

I would not air dry frozen sections after immunofluorescent staining, with 
practice and even a tiny drop of Hard Set Vectashield or Prolong Gold, you 
can get excellent coverage without bubbles.

You can always check for freezing artifact caused by too slow a freezing 
method,   A simple H&E stain on a frozen section from your block will tell 
you if you have poor snap freezing technic and getting freezing 
artiface.  As for sectioning success, that depends on what tissue, the 
temperature at which you section it, thickness, sharpness of blade or 
knife, fixed tissue with cryoprotection.  You did not elaborate on any of 

Loss of Cy3 signal can also be caused by photobleaching during overexposure 
to excitation light during CLSM, and if you are taking a long time to 
focus, etc, look at the samples, that will occur even with Antifade reagents.

In general, it is a good idea to look at your sample on day of staining or 
very soon thereafter.  I don't understand what you mean by antibody 
redistribution?  I have not heard of this nor done it - would anyone care 
to elaborate.   We are more worried about loss of fluorescent signal by 
photobleaching but in general, these special mounting medias HELP prevent 
this but the fluorophores can still lose fluorescence over time and light 

We never go to 4C to allow Prolong Gold with DAPI to set up hard nor 
VectaShield Hard Set either.  All coverslipped sections are inside a dark 
folder, sitting at room temperature.  Our tissue sections are are totally 
stained with DAPI without any of the problems  you describe.

  At 03:21 AM 10/3/2005, you wrote:
>This board is a great resource - I hope this problem has also been
>encountered by some knowledgeable people here!
>Sometimes I see huge variation in the DAPI signal when I view my
>tissue sections under the confocal microscope, and I'm wondering if
>this can be an artifact of poor freezing, as the sections that look
>the worst tend to be the ones I had trouble sectioning and show the
>worst morphology.
>At first I thought it was a problem with my mounting medium: I ordered
>a hard-set vectashield with DAPI, and while I was waiting for the
>order I borrowed a bottle of Prolong Gold from Molecular Probes. The
>Prolong Gold worked fine, I just had to seal the coverslips with nail
>polish to prevent damage to the sections. When the vectashield
>hard-set arrived my first sections showed really inconsistent DAPI
>staining - some regions nice & bright, others really weak. Generally
>it the the outside edge of the section that is well stained, and the
>interior faint. I thought perhaps the polymerizing of the mounting
>medium happened too quickly, so that it would harden around the
>tissue, which was still a bit wet from the last wash, and not
>distribute evenly. So next time I air dried the slides really well
>before mounting, but then had problems with air bubbles getting
>trapped in the mounting medium, and still not good staining into the
>middle of the sections.
>I talked to the Vectashield rep, who didn't have any good suggestions,
>but did replace the bottle with a normal non-polymerizing DAPI medium.
>But the problem of seeing DAPI staining only at the edges of the
>sections is getting worse, and have since had the same problem with
>the Prolong Gold too!
>The DAPI I use only as a counterstain to make the pictures look nice,
>but I wonder if the DAPI isn't evenly distributed, then perhaps the
>flourescence protectant is also not evenly distributed, which would
>undermine my quantitative analysis of the Cy3 signal I'm looking at.
>Yesterday's slides were the worst of all - not only was there almost
>no DAPI signal (expect a very tiny margin around each section), but
>the Cy3 signal was almost invisible too, hence the concern about the
>fluoroprotectant. I usually wait 2 days before analysing my slides,
>as the antibody seems to continue to redistribute itself, giving
>lower backgound over a day or 2, so I need to know that the
>fluorescent signal is protected. I used to put the slides immediately
>at -20C after mounting, but with the hard set I had to leave them at
>4C so it could polymerize properly.
>Help! Has anyone else dealt with this problem? What is the general
>wisdom regarding drying (or not) the slides before mounting? Does it
>have any effect? The consistency of my staining has been steadily
>decreasing with the morphology of my sections - I hope I can solve
>both problems soon!
>Thanks very much.
>Institut Curie, Paris
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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