Semi-Commercial Post; RE: autofluorescence in liver tissue

[Jim Mansfield]

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Hi all,

If chemical methods for reducing autofluorescence in FFPE tissues are
unsuccessful or insufficient (which, unfortunately, is often the case), then
there are two other approaches one might use:

1) Multispectral imaging and unmixing. This has been shown in many hundreds
of publications to reduce autofluorescence by as much as 100-fold, both for
microscopy and FFPE tissues and in small animal fluorescence imaging. A few
example citations are:

Tam, et al "Improved in vivo whole-animal detection limits of green
fluorescent protein-expressing tumor lines by spectral fluorescence imaging"
Molecular Imaging, 6, 2007, 269.

Mansfield, et al "Visualization of microscopy-based spectral imaging data
from multi-label tissue sections" Current Protocols in Molecular Biology,
2008 Oct;Chapter 14:Unit 14.19. doi: 10.1002/0471142727.mb1419s84.

Levenson, et al "Multispectral imaging in biology and medicine: slices of
life." Cytometry A. 2006 Aug 1;69(8):748-58.

2) Signal amplification. There are several HRP-based methods (such as TSA
and TSA Plus) and other amplification methods which might help. A few
citations are:

McKay, et al "Amplification of fluorescent in situ hybridisation signals in
formalin fixed paraffin wax embedded sections of colon tumour using
biotinylated tyramide." Mol Pathol. 1997 December; 50(6): 322-325.

Mehta, et al "Iron Is a Sensitive Biomarker for Inflammation in Multiple
Sclerosis Lesions" PLoS One. 2013; 8(3): e57573.


Best regards,

Jim


--

Jim Mansfield
[hidden email]
www.jmansfield.com
[phone]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Kilgore, Jason
Sent: Tuesday, June 04, 2013 5:23 PM
To: [hidden email]
Subject: Re: autofluorescence in liver tissue

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
Home of: "Microscopy and Imaging Resources on the WWW"

[George McNamara]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Jim,

Thanks for the reminder.

Hi again listserv,

Many types of autofluorescence span the entire visible region. A
microscope equipped with a Cyan Fluorescent Protein (CFP) filter set
whose emission band stops before 500 nm (ex458, em 470-495nm) will
capture "autofluorescence only". Many confocal microscopes have 458 nm
(Argon ion) or 440 nm excitation, and emission can be set to whatever is
needed. CFP excitation and spectral emission and unmixing also possible.
Of course "CFP autofluorescence" collection is also possible in
combination with TSA that I mentioned earlier.


George

On 6/4/2013 6:19 PM, Jim Mansfield wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> If chemical methods for reducing autofluorescence in FFPE tissues are
> unsuccessful or insufficient (which, unfortunately, is often the case), then
> there are two other approaches one might use:
>
> 1) Multispectral imaging and unmixing. This has been shown in many hundreds
> of publications to reduce autofluorescence by as much as 100-fold, both for
> microscopy and FFPE tissues and in small animal fluorescence imaging. A few
> example citations are:
>
> Tam, et al "Improved in vivo whole-animal detection limits of green
> fluorescent protein-expressing tumor lines by spectral fluorescence imaging"
> Molecular Imaging, 6, 2007, 269.
>
> Mansfield, et al "Visualization of microscopy-based spectral imaging data
> from multi-label tissue sections" Current Protocols in Molecular Biology,
> 2008 Oct;Chapter 14:Unit 14.19. doi: 10.1002/0471142727.mb1419s84.
>
> Levenson, et al "Multispectral imaging in biology and medicine: slices of
> life." Cytometry A. 2006 Aug 1;69(8):748-58.
>
> 2) Signal amplification. There are several HRP-based methods (such as TSA
> and TSA Plus) and other amplification methods which might help. A few
> citations are:
>
> McKay, et al "Amplification of fluorescent in situ hybridisation signals in
> formalin fixed paraffin wax embedded sections of colon tumour using
> biotinylated tyramide." Mol Pathol. 1997 December; 50(6): 322-325.
>
> Mehta, et al "Iron Is a Sensitive Biomarker for Inflammation in Multiple
> Sclerosis Lesions" PLoS One. 2013; 8(3): e57573.
>
>
> Best regards,
>
> Jim
>
>
> --
>
> Jim Mansfield
> [hidden email]
> www.jmansfield.com
> [phone]
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Kilgore, Jason
> Sent: Tuesday, June 04, 2013 5:23 PM
> To: [hidden email]
> Subject: Re: autofluorescence in liver tissue
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
> Cellular&  Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054