[Histonet] autofluorescence experiment

[Nicole Collette]

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