Re: dystrophin

From:Gayle Callis

You wrote:  

I have a neat dilution on my dystrophin  1 and 3( novocastra) and after
alexa 594, the flourescent stain was excellent however after three days  or
a week the stain starts to fade out. My dystrophin 2 in 1:3 dilution in PBS
remains the same(meaning the stains still looks excellent). Can somebody
help me figure out how can i remedy this.  I hope i can find the answer by
diluting it with PBS but the stain is not sharp enough. would it be on my
rinse?  Thank you for your help. 

Alexa dyes ARE sensitive to light, hopefully you did your staining in a
light protected box of some sort and stored stained slides in a dark place,
slide box or slide folder protected from light.  We also dilute our Alexa's
in PBS, not using anything else ie Tween 20 or protein carriers.  
Molecular Probes has several mounting medias that help with fading and can
be used with Alexa dyes to help with fading problems, this will be on their
website with a chart telling which media works best.   

It is a good idea to take photos the first time you look at them to
document results as with many fluorescent markers. Alexa dyes are more
resistant to UV exposure and quenching than FITC but not totally immune
from fading after they have been excited by UV light. 

Also, the diluted Alexa's lose fluorescence if you are not careful to
protect this reagent from dark, the one year expiration date is critical,
or so we find in our hands with the stock reagent. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (voice mail)
406 994-4303 (FAX)


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