🚀 go-pugleaf

Dear list members,

      In following the discussion of the loss of GFP fluorescence, I believe
a main point is being missed.  Rather than discussing the results of the
range of brands and colours being used by the various labs (truly the realm
of voodoo), we should consider what is causing the loss of fluoresecence.  I
firmly believe that the solvent system of the nail polish (ethyl
acetate/acetone) is responsible.  However not by directly affecting the GFP
molecules, but rather by killing the live cells being examined.  I base this
conclusion on work I did with live epidermal peels (plant material) mounted
in chamber slides.  If the coverslip forming the bottom of the chamber was
newly cemented (using nail polish) the cells would quickly be killed.
However if the coverslip was cemented and then left to outgas overnight at
room temp or at 37C the chamber could be used with no ill effects.

        In a related matter, GFP seemed to retain its fluorescence without
any trouble after paraformaldhyde fixation (4% in buffer, 1 hour fixation).
When working  reseach groups who studied yeasts, they suggested fixation in
70-100% ethanol or in acetone.  This caused immediate loss of GFP
fluorescence which I believe resulted from denaturing of the GFP molecule.
When the yeast cells were fixed in 4% paraformaldhyde the GFP fluorescence
remained strong.

Cheers,

Owen M. Schwartz
Head, Biological Imaging Facility
NIAID, NIH
Building 4, Room 424
4 Center Drive MSC 0485
Bethesda, MD  20892-0485

Phone 301-402-5883
Fax 301-402-0366
email OSchwartz@NIAID.NIH.GOV