Re: autofluorescence in liver tissue

Posted by mcammer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/autofluorescence-in-liver-tissue-tp7580412p7580421.html

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As per http://cammer.net/historical/aif/instructions/immunofluor/protocol01.htm
II.  REDUCE BACKGROUND
Incubate with NaIO4 (21.4 mg/ml in PBS) 15 min. followed by PBS wash
Alternative bleaching solutions can be fresh NaBH4 or glycine followed by PBS wash

As per http://cammer.net/historical/aif/instructions/immunofluor/troubleshoot.htm
Quench after fixation and before staining.  Try:
1.  Incubate with NaIO4 (21.4 mg/ml in PBS) 15 min.; PBS wash.
2.  NaBH4 (10mg/ml) in PBS approx. 4 repeated washes 15 min. each; NaBH4 should be dry until immediately before use; PBS wash. [More.]
3.  Incubate with glycine; PBS wash.
Screen for autofluorescence before staining.

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

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From: Confocal Microscopy List [[hidden email]] on behalf of Armen Khatchadourian [[hidden email]]
Sent: Tuesday, June 04, 2013 5:42 PM
To: [hidden email]
Subject: Re: autofluorescence in liver tissue

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I had a similar problem with paraffin-embedded pancreatic sections. But I was getting much less autofluorescence in the far red region, compared to green and red, hence I started using AlexaFluor 647 -labeled secondary antibodies. I don't know if this would work for liver though.
Best,
Armen
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From: Confocal Microscopy List [[hidden email]] On Behalf Of Kilgore, Jason [[hidden email]]
Sent: Tuesday, June 04, 2013 5:23 PM
To: [hidden email]
Subject: Re: autofluorescence in liver tissue

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Autofluorescence can be especially bad for paraffin-embedded sections.  I'm not sure liver is particularly worse, though, compared to many tissues.

I've had very good luck with sodium borohydride washing to reduce autofluorescence, particularly in the green and red range.

Another thing I've found to reduce it is to use Histoclear or other limonine solvents for deparaffinization, instead of xylenes.

Another thing to try is to amplify your label, to help overcome the background, using biotin/streptavidin, Tyramide signal amplification, or Qdots.

Hope that helps.  Cheers,

Jason


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Tuesday, June 04, 2013 10:24 AM
To: [hidden email]
Subject: autofluorescence in liver tissue

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I am working with a lab looking at IF stained sections of formalin-fixed, paraffin embedded pieces of mouse or rat liver.  If there was any actual staining in their first try on our confocal, it was largely washed out by the lovely and strong autofluorescence.  Because their animal experiment takes 8 weeks, it's not trivial (or inexpensive) to get new tissue, so they have been using existing blocks.  No one in their lab had the foresight to put up snap-frozen tissues for cryosectioning.  I have them looking at this document: http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf but I'm wondering if liver is always a problem with autofluorescence, or is there maybe something in this lab's sample prep protocol that's making it worse.  Any ideas?

Doug

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Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
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