autofluorescence in liver tissue

[Cromey, Douglas W - (dcromey)]

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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

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
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"

[Kilgore, Jason-2]

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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

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
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"

[Mark A. Sanders]

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Hi Doug,

The liver autofluorescence may be due to lipofuscin.  Try treating with Sudan Black.  Sudan Black binds lipophilic compartments and, being black, absorbs the excitation energy and quenches lipofuscin.  

Here is a paper:

Schnell SA, Staines WA, Wessendorf MW.  Reduction of lipofuscin-like
autofluorescence in fluorescently labeled tissue.  J Histochem Cytochem.
1999 47(6):719-30.


Cheers,
Mark
****************************************************
Mark A. Sanders      University of Minnesota
Program Director      Twin Cities Campus
University Imaging Centers      
St. Paul office ph:  612-624-3454
Mpls office ph:  612-626-3645
fax: [phone]
www.uic.umn.edu


 

On Jun 4, 2013, at 12:23 PM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:

> *****
> 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"

[Armen Khatchadourian]

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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
________________________________________
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

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
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"

[Martin Wessendorf-2]

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Dear Doug--

On 6/4/2013 12:23 PM, Cromey, Douglas W - (dcromey) wrote:

> 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?

Several questions:

0)  Are you sure it's autofluorescence and not a problem with your
staining protocol?  Do you see the same autofluorescence in sections
that haven't been stained?
1)  What does the autofluorescence look like?  Is it small, bright dots
or a uniform high background?
2)  Can you see it with the fluorescein filter?  With the rhodamine
filter?  With UV excitation?
3)  What color is the autofluorescence under a wide-band UV filter?

The cure depends on the cause.  If you see a uniform high background,
it's probably due to fixation, and treating the sections with sodium
borohydride (NaBH4) as Jason Kilgore suggested should help.  See:
http://www.ncbi.nlm.nih.gov/pubmed/9765122

If the autofluorescence looks like small, bright dots that don't
photobleach, show up using all your filter sets and look orange using a
wide-band UV filter, it's probably lipofuscin.  That can be reduced by
treating with Sudan Black, as Mark Sanders suggested, or with CuSO4,
which is described in the same paper.  CuSO4 is the better choice if
you're running your tissue through xylene after staining--the Sudan
Black will be eluted by lipophilic solvents.

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[mcammer]

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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]

________________________________________
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
________________________________________
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

*****
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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Hi Doug,

Tyramide signal amplification, with 11 washes after the primary, 11
washes after the 2ndAb-HRP (need to re-titer the antibodies compared to
other methods ... and keep the tissue submerged at all times - my thanks
to Richard Burry for the tip - see his Immunocytochemistry book). For
killing the HRP between rounds (if multicolor TSA is needed) see
peroxAbolish info in

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant
tissue using laser scanning cytometry.
<http://www.ncbi.nlm.nih.gov/pubmed/21929847>

*Takahashi* H, Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R,
Barker S, Burke GW, Tzakis AG, *Ichii* H.

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub
2011 Sep 16.

PMID:
    21929847


You are not required to use the kit (Mol Probes or PerkinElmer sell TSA
kits) HRP - or other components (except the fluorescent tyramide of
course). I still have not tried it (not needed so far), but am intrigued
by the HRP80 at
http://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html 


If anyone has experience - either good or bad - with HRP80 or HRP50 or
HRP20 - please let us know here on the listserv or tell me privately.

George
p.s. with respect to HRP80 * TSA ... positive specimens might need a
warning: "do not look through the eyepiece with remaining eye" (yes,
that's a joke - eyesight should recover after a few minutes). If you
don't do enough washes, everything will be blindingly bright. Also,
liver may have endogenous biotin sticking out, so need to make sure to
block well (and always do the controls) when using streptavidin
conjugates like HRP80.


On 6/4/2013 12:23 PM, Cromey, Douglas W - (dcromey) wrote:

> *****
> 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

[Csúcs Gábor-2]

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Dear All,

A potential new user would have interest in using the VT-Hawk confocal
system from Visitech. Is there anyone who has/had experience with the
system? On paper the specifications look very nice, but I'd really
appreciate your comments (also privately if required).

Thanks     Gabor

Light Microscopy and Screening Centre
ETH Zurich, HPM G41
Schafmattstrasse 18
CH-8093, Zurich
Switzerland

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Mobile: [phone]