[Histonet] Nail Polish sealant

[gayle callis]

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?

 

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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

HTL/HT/MT(ASCP)

Bozeman MT