[Histonet] autofluorescence experiment

[John Kiernan]

I don't know any argument against using both copper sulphate and Sudan black B, and you have shown that the combination reduces the autofluorescence of bone. It's interesting that both are applied after immunofluorescent staining and are reported to cause some  reduction of the desired fluorescence (Schnell et al 1999 J. Histochem. Cytochem. 47:719-730; Baschong et al 2001 J. Histochem. Cytochem. 449:1565-1571). 
 
Earlier chemical methods for suppressing autofluorescence involved pre-treatment of sections with either a heavy metal compound or a non-fluorescent dye. 0.2% osmium tetroxide for 5 mins is very effective; needs hours in slowly running tap water to wash it out before fluorescent staining. The dye Direct blue 1 (CI 24410), also known as Chicago sky blue B, Niagara blue 6B and pontamine sky blue, was recommended by Cowen et al 1985 Histochemistry 82:205-208 and  Kutvolgyi et al 2006 Biotech. Histochem. 81:4-6.  Cowen et al used a 0.05% solution of the dye in PBS with 1% DMSO. I haven't treid it myself. 
 
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: Nicole Collette <collette2 <@t> mail.llnl.gov>
Date: Wednesday, February 3, 2010 19:12
Subject: [Histonet] autofluorescence experiment
To: Histonet <@t> lists.utsouthwestern.edu

> Hello, All,
> 
> I am working on doing some IF stains with bone samples (lucky 
> me!). I 
> am having a difficult time sometimes to assess the antibody 
> since the 
> autofluorescence gets in the way. I am using undecalcified, FFPE 
> sections (late embryo and neonate mouse bones). Without 
> treatment, I 
> see autofluorescence everywhere, but most frustrating is the red 
> blood cells and the mineralized matrix. It took me a while to 
> get 
> samples that are properly fixed, section well, and stay on the 
> slides, so I am not particularly jazzed about messing with the 
> fixation protocol. Thus, I conducted an experiment today of 
> several 
> published and voodoo methods to reduce autofluorescence with 
> samples 
> that did NOT undergo the IF protocol :
> 
> no treatment
> photobleaching, fluorescent light box, up to 2 weeks
> photobleaching, UV crosslinking light, 2 inches from source, 24h
> 10mM copper sulfate in 50mM ammonium acetate, pH 5.0
> 0.25% (v/v) NH3 in 70% ethanol (in water)
> 0.25% (v/v) NH4OH in 70% ethanol (in water) (since it was 
> unclear 
> from the published reference what the source of ammonia was, and 
> I 
> have made this mistake before on some other thing)
> 0.3% (w/v) Sudan Black in 70% ethanol (in water)
> 
> I found that the most effective treatment in my hands is Sudan 
> Black 
> for cell-based autofluorescence, but it did not seem to impact 
> the 
> autofluorescence from the mineralized matrix at all, while 
> copper 
> sulfate had significant impact on the autofluorescence in 
> mineralized 
> bone, but did not quench cell-based autofluorescence as well as 
> the 
> Sudan Black (it had an even but incomplete impact over the 
> entire 
> section). I have tried Sudan Black on my slides before, and have 
> found that it did not seem to interfere significantly with the 
> antibody signal. I modified the Sudan Black protocol to 
> eliminate the 
> "goopies" and "chunkies" resulting on my slides from previous 
> attempts, and am happy with the results- a light, even stain.
> 
> My question to all you chemistry folks: Is there some reason why 
> copper sulfate treatment followed by washing and subsequent 
> Sudan 
> Black treatment (and more washing) cannot or should not be used? 
> I 
> tried them together on my unstained slides, they look pretty 
> darn 
> fabulous. Just striving for clean data and beautiful pictures.
> 
> Thanks again for all your help,
> Sincerely,
> 
> Nicole Collette
> Lawrence Livermore National Lab/ UC Berkeley
> 
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