[Histonet] H2DCFDA fading, photobleaching and antifade mounting medias

[Gayle Callis]

Glenn,  You wrote:

I am using H2DCFDA for the detection of cellular reactive oxygen species 
following treatment with various agents. Cells are subsequently mounted in 
anti-fadent with DAPI (Slow Fade Gold with DAPI).

   The green fluorescence appears awfully weak. I have noticed this in past 
experiments looking at cytochrome c release with FITC detection.  Could the 
antifadent be the culprit? Don't some antifadent products quench 
fluorescein right-off-the-bat? Would it be best to just do a standard 
aqueous mount and be extra careful with light exposure?

Reply:

What you are experiencing is NOT quenching but photobleaching.  FITC is far 
easier to photobleach when exposed to UV excitation and not performing the 
staining in the dark (under cover!) The mounting media helps prevent 
photobleaching but not 100%, however if you use Alexa 488 from Molecular 
Probes instead of FITC as the fluorophore, you will probably have far 
better retention of fluorescnce with the Alexa's.  It definitely will be 
brighter and you can look at it longer with UV light.

It is not only mounting media but also the choice of fluorophore. There are 
several mounting medias that Molecular Probes did a comparison on  and 
showed(results available online)which mounting medias worked better for 
fluoresecence.

You can learn a great deal about fluorsceinated fluorophores (FITC, TRITC, 
Texas Red) versus Alexa dyes that are sulfonated with the latter be 
superior.  These are more more resistant to photobleaching and definitely 
worth a try.  Molecular Probes Handbook explains this, although it is a 
rather "physical chemistry" oriented subject.  The original publication in 
J HIstochem Cytochem by the Hauglands who developed the use of Alexa dyes 
is a good read.

Microscopy Today just had a wonderful comparison of mounting medias for IFA 
by Collins, go back in Histonet Archives to access the website for this 
free article.

I have never had a mounting media fade FITC immediately, but I do enjoy 
good fluroescent signal retention with this after using Molecular Probes 
hard set Prolong Gold ready to use with or without DAPI. I have use 
AquaMount, but examination of slide is same day - photobleaching generally 
occurs more at time of UV light excitation so we take photos at time of 
viewing sections.

There are some ways to increase the signal - we use Strepavidin-Alex dyes 
with biotinylated secondary or a biotinylated primary to amplify more -

Hope this helps.



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