[Histonet] Deterioration rate of fluors used in IF staining

Gayle Callis gayle.callis <@t> bresnan.net
Sat Jan 12 16:21:44 CST 2008

We now coverslip with Molecular Probes Prolong Gold antifade reagent, with 
or without DAPI.  We have found some antifade mounting medias cause fading 
if left overnight, but if the slides are examined right after staining, 
fading has not occured.  So be careful about what antifade mounting medias 
you use.  Also, some fluorophores do not withstand mounting medias and will 
fade right away.  I believe Cy2 is one, and also Alexa 630 - Jackson and 
Molecular Probes provide this information.  We have found RRX (rhodamine red 
extra) from Jackson Immunorearch, is more reisistant to staining than TRITC, 
both are derivatives from fluorescein but have different chemical 

We don't bother to refrigerate but do keep the sections in the dark and try 
to view them the next day after staining,coverslipping.    Selection of 
fluorophore is important, and Alexa dyes are more resistant to fading and 
also much brighter than fluorescein related fluorophores (FITC, Rhodamine, 
Texas Red).  It is important to learn about the fluorophores and their 
chemstry.  The experts on this are Invitrogen/Molecular Probes.  Alexa 488 
dye will not fade as fast as FITC, but it will fade eventually - one can 
always test this by staining several sections and pulling one a day until 
the fluorophore has faded.

All staining with fluorophore steps are performed under cover of a black 
towel to protect from any light, and when making up the reagents, keeping 
the diluted fluorophore conjugates in a drawer before application is 
important.  It isn't the cold that prevents fading, it is lessening the 
exposure to light.  Most of the time, refrigerators are closed with the 
lights off, and slides are kept in the dark in a cardboard flat/folder, 
essentially protected from the light until examination.  We have put slides 
in a drawer if we can't examine them the same or next day.  We do store 
fluorophore conjugated reagents according to manufacturer instructions, in a 
lidded, dark box, in a refrigerator or freezer in a light protected box.

Gayle M. Callis
Bozeman MT 59715

----- Original Message ----- 
From: "Sally Price" <sprice2003 <@t> gmail.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, January 11, 2008 3:37 PM
Subject: [Histonet] Deterioration rate of fluors used in IF staining

> Netters:
> I realize that the fluorescnt dyes like Alexa/Dylight deteriorate over 
> time,
> but I was wondering: Just how fast?  For years we've been told to protect
> reagents from direct light during the staining procedure and to keep 
> stained
> slides in the dark when not under the scope, but how critical is this --  
> and
> does keeping the reagents/slides refrigerated slow down the process, or am 
> I
> just wasting the effort?  Maybe some vendors can chime in here too.
> Cheers!
> -- 
> Sally Price
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