releasing old histology stains (which are fluorescing in red)

[Holly L. AARON]

Dear List - Not strictly a confocal question, but...

Does anyone have a protocol for releasing old histology stains from parafin embedded tissue sections? I have users who are having a hard time getting around a lot of red background in their samples. They have already removed the parafin and have been able to release some of the stains using high or low pH baths, but still see a significant signal in the red channel. They want to do multi-color antibody labeling on the sections now. They can do it avoiding red, using green and blue and maybe a far-red (we have not characterized the spectra yet), but if there is a way to release these old stains, it would be very useful for them. They think the only stains on the tissue are H&E (which they think is not the problem as they have done this before, although eosin is brightly red?) and PAS-aurantia.

Thanks for any tips or ideas!

[Cameron Nowell]

Hi Holly,
 
you should be able to leach out the eosin with alchohol. I am not sure that your antigens are going to be very healthy after various treatments with alcohol, high and low pH buffers etc.
 
The other choice would be to bleach the eosin out with your mercury lamp. So it will still be there, just not fluorescent.
 
 
Good luck
 
 
Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Holly Aaron
Sent: Fri 31/07/2009 7:26 AM
To: [hidden email]
Subject: releasing old histology stains (which are fluorescing in red)


Dear List - Not strictly a confocal question, but...

Does anyone have a protocol for releasing old histology stains from parafin embedded tissue sections? I have users who are having a hard time getting around a lot of red background in their samples. They have already removed the parafin and have been able to release some of the stains using high or low pH baths, but still see a significant signal in the red channel. They want to do multi-color antibody labeling on the sections now. They can do it avoiding red, using green and blue and maybe a far-red (we have not characterized the spectra yet), but if there is a way to release these old stains, it would be very useful for them. They think the only stains on the tissue are H&E (which they think is not the problem as they have done this before, although eosin is brightly red?) and PAS-aurantia.

Thanks for any tips or ideas!


--


Holly L Aaron

Molecular Imaging Center

Cancer Research Laboratory

251 Life Sciences Addition #2751

Berkeley, CA  94720-2751

510.642.2901

510.642.5741 FAX

[hidden email]

http://imaging.berkeley.edu <http://imaging.berkeley.edu/>

 

[Diane G Miller]

Hi Holly,

 

If they use a weak solution of acetic acid in water.  .05% that should decolorize the slide.

 

Hope that helps.

Diane

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]On Behalf Of Holly Aaron
Sent: Thursday, July 30, 2009 2:26 PM
To: [hidden email]
Subject: releasing old histology stains (which are fluorescing in red)

Dear List - Not strictly a confocal question, but...

Does anyone have a protocol for releasing old histology stains from parafin embedded tissue sections? I have users who are having a hard time getting around a lot of red background in their samples. They have already removed the parafin and have been able to release some of the stains using high or low pH baths, but still see a significant signal in the red channel. They want to do multi-color antibody labeling on the sections now. They can do it avoiding red, using green and blue and maybe a far-red (we have not characterized the spectra yet), but if there is a way to release these old stains, it would be very useful for them. They think the only stains on the tissue are H&E (which they think is not the problem as they have done this before, although eosin is brightly red?) and PAS-aurantia.

Thanks for any tips or ideas!

[Lachlan Walsh]

Hi Holly,

If it's a leukobasic fuchsin then 1% sodium borohydride should reduce it
and make it colourless. In the process you'll get rid of any aldehyde
fixation induced autofluorescence, which can only be a good thing.

Lachlan

Diane G Miller wrote:

> Hi Holly,
>  
> If they use a weak solution of acetic acid in water.  .05% that should
> decolorize the slide.
>  
> Hope that helps.
> Diane
>
>     -----Original Message-----
>     *From:* Confocal Microscopy List
>     [mailto:[hidden email]]*On Behalf Of *Holly Aaron
>     *Sent:* Thursday, July 30, 2009 2:26 PM
>     *To:* [hidden email]
>     *Subject:* releasing old histology stains (which are fluorescing
>     in red)
>
>     Dear List - Not strictly a confocal question, but...
>
>     Does anyone have a protocol for releasing old histology stains
>     from parafin embedded tissue sections? I have users who are having
>     a hard time getting around a lot of red background in their
>     samples. They have already removed the parafin and have been able
>     to release some of the stains using high or low pH baths, but
>     still see a significant signal in the red channel. They want to do
>     multi-color antibody labeling on the sections now. They can do it
>     avoiding red, using green and blue and maybe a far-red (we have
>     not characterized the spectra yet), but if there is a way to
>     release these old stains, it would be very useful for them. They
>     think the only stains on the tissue are H&E (which they think is
>     not the problem as they have done this before, although eosin is
>     brightly red?) and PAS-aurantia.
>
>     Thanks for any tips or ideas!
>
>     --
>
>     Holly L Aaron
>
>     Molecular Imaging Center
>
>     Cancer Research Laboratory
>
>     251 Life Sciences Addition #2751
>
>     Berkeley, CA  94720-2751
>
>     510.642.2901
>
>     510.642.5741 FAX
>
>     [hidden email] <mailto:[hidden email]>
>
>     http://imaging.berkeley.edu
>
>      
>

[JOEL B. SHEFFIELD]

The red certainly seems to be from Eosin.  We use old H&E slides to illustrate fluorescence of goblet cells in intestinal sections.  I'm not sure what will take it out --Eosin is soluble in alcohol, but I'm not sure how it binds to the cellular components.  You might try other organic solvents.

Joel

On Thu, Jul 30, 2009 at 5:26 PM, Holly Aaron <[hidden email]> wrote:

Dear List - Not strictly a confocal question, but...

Does anyone have a protocol for releasing old histology stains from parafin embedded tissue sections? I have users who are having a hard time getting around a lot of red background in their samples. They have already removed the parafin and have been able to release some of the stains using high or low pH baths, but still see a significant signal in the red channel. They want to do multi-color antibody labeling on the sections now. They can do it avoiding red, using green and blue and maybe a far-red (we have not characterized the spectra yet), but if there is a way to release these old stains, it would be very useful for them. They think the only stains on the tissue are H&E (which they think is not the problem as they have done this before, although eosin is brightly red?) and PAS-aurantia.

Thanks for any tips or ideas!

--

Holly L Aaron

Molecular Imaging Center

Cancer Research Laboratory

251 Life Sciences Addition #2751

Berkeley, CA  94720-2751

510.642.2901

510.642.5741 FAX

[hidden email]

http://imaging.berkeley.edu

 

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Russell Spear]

Hi

You might try a freshly made solution of sodium boron hydride 0.5 - 1.0%
or perhaps hydrogen peroxide 1-2% in the former case use in the hood as
H2 is the gas is formed and in neither case seal the containers as
pressure can build up.  About 30 mins to 1 hr.  It shouldn't damage the
antigenic sites as I use it to decrease autofluorescence in FISH &
antibody labeled specimens.

Russ


Russell N. Spear
Sr. Research Specialist
Dept. of Plant Pathology
Univ. of Wisconsin
1630 Linden Dr.
Madison WI 53706

voice 608.263.2093
fax     [phone]
>>> "JOEL B. SHEFFIELD" <[hidden email]> 07/31/09 8:57 AM >>>
The red certainly seems to be from Eosin.  We use old H&E slides to
illustrate fluorescence of goblet cells in intestinal sections.  I'm not
sure what will take it out --Eosin is soluble in alcohol, but I'm not
sure
how it binds to the cellular components.  You might try other organic
solvents.

Joel


On Thu, Jul 30, 2009 at 5:26 PM, Holly Aaron <[hidden email]>
wrote:

>  Dear List - Not strictly a confocal question, but...
>
> Does anyone have a protocol for releasing old histology stains from
parafin
> embedded tissue sections? I have users who are having a hard time
getting
> around a lot of red background in their samples. They have already
removed
> the parafin and have been able to release some of the stains using
high or
> low pH baths, but still see a significant signal in the red channel.
They
> want to do multi-color antibody labeling on the sections now. They can
do it
> avoiding red, using green and blue and maybe a far-red (we have not
> characterized the spectra yet), but if there is a way to release these
old
> stains, it would be very useful for them. They think the only stains
on the
> tissue are H&E (which they think is not the problem as they have done
this

> before, although eosin is brightly red?) and PAS-aurantia.
>
> Thanks for any tips or ideas!
>
>  --
>
> Holly L Aaron
>
> Molecular Imaging Center
>
> Cancer Research Laboratory
>
> 251 Life Sciences Addition #2751
>
> Berkeley, CA  94720-2751
>
> 510.642.2901
>
> 510.642.5741 FAX
>
> [hidden email]
>
> http://imaging.berkeley.edu
>
>
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Francisco J H Blazquez]

I am wrong or eosin shows green fluorescence? I remember that eosin is
used to show apoptotic bodies and its fluorescence is green.

Cameron Nowell escreveu:

> Hi Holly,
>  
> you should be able to leach out the eosin with alchohol. I am not sure that your antigens are going to be very healthy after various treatments with alcohol, high and low pH buffers etc.
>  
> The other choice would be to bleach the eosin out with your mercury lamp. So it will still be there, just not fluorescent.
>  
>  
> Good luck
>  
>  
> Cam
>  
>  
>  
> Cameron J. Nowell
> Microscpy Manager
> Central Resource for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>  
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
>  
> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>  
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Holly Aaron
> Sent: Fri 31/07/2009 7:26 AM
> To: [hidden email]
> Subject: releasing old histology stains (which are fluorescing in red)
>
>
> Dear List - Not strictly a confocal question, but...
>
> Does anyone have a protocol for releasing old histology stains from parafin embedded tissue sections? I have users who are having a hard time getting around a lot of red background in their samples. They have already removed the parafin and have been able to release some of the stains using high or low pH baths, but still see a significant signal in the red channel. They want to do multi-color antibody labeling on the sections now. They can do it avoiding red, using green and blue and maybe a far-red (we have not characterized the spectra yet), but if there is a way to release these old stains, it would be very useful for them. They think the only stains on the tissue are H&E (which they think is not the problem as they have done this before, although eosin is brightly red?) and PAS-aurantia.
>
> Thanks for any tips or ideas!
>
>
>  

--
Prof. Dr. Francisco Javier Hernandez Blazquez
Universidade de São Paulo
Fac. de Medicina Veterinária e Zootecnia
Departamento de Cirurgia - Setor de Anatomia
Av. Prof. Dr. Orlando Marques de Paiva, 87
05508-270 - São Paulo (SP) - Brasil
http://www.fmvz.usp.br/index.php/site/docentes/lista_de_docentes/francisco_javier_hernandez_blazquez
Tel..[phone]  Fax  [phone]
email: [hidden email]

[Koo, Lily (NIH/NIAID) [E]]

Dear List,

 

We wonder what is everyone’s experience with the photoconversion of YFP into a CFP-like emission spectra during acceptor photobleaching FRET?  There was a correspondence article in Nature Methods in 2005 citing this phenomenon, a year later it was refuted, and was confirmed again in 2007.  I haven’t come across too many FRET paper discussing it and wonder if it is really an established observation?

 

Thanks,

 

http://www.nature.com/nmeth/journal/v2/n11/full/nmeth1105-801.html

 

Lily

 

 

[Zoltan]

 Hi Lily,

  We are also aware of the potential artifact that comes from bleaching ypf and then exciting cfp with 405.  We do get a very strong (artificial) signal in the donor channel when using 405 nm even in  a control prep where there is no donor at all!  It seems to be a conversion of the yfp molecules to a form that can be easily excited by 405, and also (but not nearly as much) by 458 nm.  Our recipe now is to excite cfp with 458. 

  I hope this helps,

 

Zoltan

On Tue, Aug 4, 2009 at 8:40 PM, Koo, Lily (NIH/NIAID) [E] <[hidden email]> wrote:

Dear List,

 

We wonder what is everyone’s experience with the photoconversion of YFP into a CFP-like emission spectra during acceptor photobleaching FRET?  There was a correspondence article in Nature Methods in 2005 citing this phenomenon, a year later it was refuted, and was confirmed again in 2007.  I haven’t come across too many FRET paper discussing it and wonder if it is really an established observation?

 

Thanks,

 

http://www.nature.com/nmeth/journal/v2/n11/full/nmeth1105-801.html

 

Lily

 

 

--

Zoltan Cseresnyes
Facility manager, Imaging Suite