[Histonet] Nail Polish sealant
leiker <@t> buffalo.edu
Fri Jan 13 08:40:12 CST 2012
Interesting. I've never had an issue.
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Andrea Hooper
Sent: Thursday, January 12, 2012 10:04 PM
To: gayle callis
Cc: Histonet; <kasaim <@t> mail.nih.gov>
Subject: Re: [Histonet] Nail Polish sealant
Amen Gayle, as always you hit the nail on the head. I never use nail polish sealant because of being burned so many times due to the isopropanol leaching into - and ruining - the sample. In addition if it gets onto pricey fluorescence lenses it's difficult to clean. It's not worth the risk, especially given the great hard set mountants available. I also detest the mountants with DAPI mixed in. Nothing better tan doing your own even counter stain with Hoechst 33342 for 10 minutes before mounting for crisp beautiful nuclei.
On Jan 5, 2012, at 12:35 PM, "gayle callis" <gayle.callis <@t> bresnan.net> wrote:
> You wrote:
> We are currently starting up some IHC on frozen tissue sections.
> After staining with different fluorescent antibodies, we end with
> applying DAPI w/Prolong gold and then coverslipping. We would like to
> seal the coverslip so that we can keep the slides longer. Any
> suggestions on where and how best to apply the nail polish for a permanent fix on the coverslips?
> Prolong Gold antifade reagent is a hard seal to begin with. We only
> seal ends but not the sides of cover glass. Our coverslips go right to edge of
> slides e.g. 25 X 30, 25 x 40, or 25 X 50. We don't like having nail
> polish slop over the edges onto back of slides but one could seal all sides
> of coverslip if careful. We buy the thinner top or base coat nail
> polish, but in general, prefer to use thinned mounting media rather than
> nail polish. There can be some issues here. If you are trying to view
> GFP or RFP (red fluorescence protein) labeled cells or tissue
> components, you should not seal the coverslip with nail polish since
> the alcohol in nail polish leaches under the coverslip and causes GFP/RFP to fade. This fact
> was published in Science. We found that dumping out cheap clear nail
> polish from bottle, rinsing away the residue with acetone, and then
> adding permanent mounting media and thinning that with toluene to the
> consistency of top coat nail polish works best. Toluene or xylene
> based sealants cannot leach under the cover glass since these solvents are NOT miscible with water
> in the PBS. Thinned mounting media is better sealant for GFP purposes (no
> fading) and also works for IF stained sections (perfect seal). We
> love the little brush in the nail polish bottle for application.
> Thicker clear nail polish (for non GFP studies) or IF stained sections
> is messy during application so we buy the cheapest top coat polish we can find at Walmart.
> DAPI in the Prolong Gold will cause an uneven staining gradient so
> that some of the nuclei in the center of a section are not stained as
> brightly as the nuclei on the outer edges of a stained section. The
> cause is not getting enough thicker Prolong Gold/DAPI over the section
> or not having just the right amount of buffer on the section to permit a good flow of this
> wonderful mounting media over the section. We now complete all IF
> staining then stain with a DAPI solution before cover slipping with Prolong
> Gold. You can buy ready to use DAPI solutions from Pierce or Biogenex, or
> make up the solution in house. You can find the recipe at IHCworld website
> or simply Google.
> We do NOT store our IF stained slides in the cold, but in a folder at
> RT in a dark drawer before viewing on the day after staining to reduce any
> movement/flow under the coverslip. Fluorophores can and will eventually
> fade. I do not recall any studies saying storing IF stained slides in the
> cold reduces fading but we never have space to do cold storage anyway and
> store slides at RT. The new fluorophores (Alexas and Dylights) remain
> stable over a longer time even for several weeks compared to
> fluorescein derivatives e.g. FITC TRITC.
> Gayle M. Callis
> Bozeman MT
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
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