[Histonet] Histonetters Assemble

From:Heather.A.Harper@pcola.med.navy.mil


To me it sounds like you are having paraffin problems and not fixation.
Maybe you are not leaving your slides in the oven long enough, or the
paraffin from the embedding station has water or something else in it
creating this opaque film.

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Subject: Histonet Digest, Vol 22, Issue 29

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Today's Topics:

   1. re Hu Ab (Carl Hobbs)
   2. Let's Play solve this problem.... (Timothy Macatee)
   3. Re: Antigen retreival on mouse GFAP. (Jan Shivers)
   4. Re: antigen retrevial/ slides (Jan Shivers)
   5. Re:sectioning problems (Gayle Callis)
   6. Re: Let's Play solve this problem.... (Jackie M O'Connor)
   7. Re: Antigen retrieval on mouse GFAP (Melissa Gonzalez)
   8. Re: removing silver from counters, etc. (Gayle Callis)
   9. Re: antigen retrevial/ slides (Gayle Callis)
  10. "Hypo" is RE: [Histonet] removing silver from counters, etc.
      (Gayle Callis)
  11. Re:sectioning problems (Vinnie Della Speranza)


----------------------------------------------------------------------

Message: 1
Date: Fri, 23 Sep 2005 15:46:29 +0100
From: "Carl Hobbs" 
Subject: [Histonet] re Hu Ab
To: 
Message-ID: <007301c5c04d$956cafb0$112b5c9f@Carlos>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
	reply-type=original

I use anti Hu C/D on pwax sections of human autopsy brain, after HIER. 
Molecular Probes A-21276, around 1/3000 dilution factor. Very good results, 
suprisingly. The Ab reagent also works vwell in pwax sections of chick, 
mouse and rat.
carl 




------------------------------

Message: 2
Date: Fri, 23 Sep 2005 10:49:28 -0400
From: Timothy Macatee 
Subject: [Histonet] Let's Play solve this problem....
To: Histonetters 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Histonetters Assemble!

I'm having a recurring problem with my H & E stained slides.  The staining
is weak, especially away from the edges of the tissue.  It looks almost like
there is still an opaque paraffin haze to the tissue.  It could be a
processing problem because it occurs more towards the middle of the section.
Is something not getting into the tissue far enough?  Is it Fixative (PFA or
Formalin), or processing reagents?  Or am I just whistling dixie?

Tim Macatee
NYU Medical Center
Research Histology Core





------------------------------

Message: 3
Date: Fri, 23 Sep 2005 10:31:07 -0500
From: "Jan Shivers" 
Subject: Re: [Histonet] Antigen retreival on mouse GFAP.
To: 
Cc: histonet 
Message-ID: <001901c5c053$d143b0a0$41065486@auxs.umn.edu>
Content-Type: text/plain;	charset="iso-8859-1"

In my experience with various mammals, antigen retrieval is not needed for
GFAP IHC.  I run mine (using Dako's rabbit anti-cow GFAP) at 30 minutes
primary incubation (3 ug/ml Ab working concentration), 30 minutes
anti-rabbit IgG EnVision+/HRP, and 10 minutes AEC chromogen.

Jan Shivers
U of MN Vet Diag Lab

*
----- Original Message ----- 
From: 
To: 
Sent: Friday, September 23, 2005 5:27 AM
Subject: [Histonet] Antigen retreival on mouse GFAP.






Hi

Does anyone have any experience with using trypsin as the method of antigen
retreival when staining GFAP on mouse brains???  Any advice on
concentration, temperature and incubation time would be appreciated.

Thanks


Susan.





------------------------------

Message: 4
Date: Fri, 23 Sep 2005 10:34:12 -0500
From: "Jan Shivers" 
Subject: Re: [Histonet] antigen retrevial/ slides
To: "Steven Coakley" 
Cc: histonet 
Message-ID: <002601c5c054$3f932180$41065486@auxs.umn.edu>
Content-Type: text/plain;	charset="iso-8859-1"

Poly-L-Lysine.  Never had a problem with adherence after HIER when using
them.  If you coat your own, let them dry overnight (covered) on edge, so
excess solution can drain off and not puddle up.

Jan Shivers
U of MN Vet Diag Lab

----- Original Message ----- 
From: "Steven Coakley" 
To: 
Sent: Friday, September 23, 2005 8:41 AM
Subject: [Histonet] antigen retrevial/ slides


> Are there any recommendations for a good adhesive slide to use for
IHC/antigen retrevial.  Also method for drying these slides
>
> Steve
>
>
> ---------------------------------
> Yahoo! for Good
>  Click here to donate to the Hurricane Katrina relief effort.
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>




------------------------------

Message: 5
Date: Fri, 23 Sep 2005 09:41:53 -0600
From: Gayle Callis 
Subject: [Histonet] Re:sectioning problems
To: "Tyler W." ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050923092322.01b67910@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

First and foremost, if you are having the ribbon pulling up as chuck with 
block (Point #3) you need to make sure  knife angle setting on the 
microtome is correct.  Check your manufacturers instructions on HOW to set 
a correct knife angle for your particular microtome.  it sound as though 
the clearance angle is incorrect.  I don't think the paraffin is the 
problem, see last comments.

You did not say what tissues you are sectioning animal?  human? mouse? nor 
how the tissue is processed?  If the tissues are dehydrated, cleared and 
infiltrated with paraffin - optimally for your particular tissue? then you 
should experience good sectioning with correct microtome settings.  Beware 
of infiltrating tissues with excessively hot paraffin, 60C is all that we 
allow with all your animal tisues, over that - I think and with long 
infiltration times, you dry out and create some sectioning problems.

64C is pretty hot in my estimation for embedding, with ANY paraffin.  We 
set ours at 60C only.  You have hot and cold surfaces on your embedding 
center to help you out, these are usually preset at factory although we can 
change that with our Sakura embedding center.  Too hot is an enemy, as you 
cook the tissues even more.  Even the kind of blades you use will affect 
how your sectioning goes, and we all have our favorites - be sure to try 
others on the market!!!

Dirrerent brand paraffins will have different sectioning qualities 
depending on what additives they add to make sectioning easier, etc.  Some 
paraffins are sticky, others are harder, some you have more compression, 
but with care and correct knife angle setting, one can usually learn to 
work with ANY paraffin.   These additives will cause the paraffins to look 
different too - opacity, clarity, hardness, ribboning ability, tackiness as 
you have noticed.

Although I like flowers, I favor the brownies!!!





At 11:20 PM 9/22/2005, you wrote:
>I recently have been having problems pulling ribbons from my microtome and 
>was
>wondering if anyone has advice.
>
>1.) I was having problems pulling ribbons from newly embeded blocks, but 
>ones that had
>been embeded previously were very easy to pull ribbons from.  The new ones 
>would simply
>curl on the blade and wouldn't even grab on to the adjascent sections.
>
>2.) I changed the paraffin in the embedding station because I noticed all 
>the temeratures
>were set to 64 C when the melting point for the Surgipath paraffin formula 
>'R' was 56-67C.
>Could this have been a problem?
>
>3.) I switched to an old lot of Fisher embeding medium and now all the 
>section come off, but
>on occasion when the chuck on the mictomome comes back up it seems to (via 
>static
>electricity?) pull the ribbon from the microtome blade and interupt the 
>ribbon.
>
>4.) Also, visually the Fisher paraffin blocks look almost transparent 
>whereas the Surgipath
>blocks seem opaque and white.
>
>I'm relatively new to the histology game and I realize that there is both 
>art and science
>involved, but this is clearly unacceptable behavior from the paraffin, 
>something is surely
>wrong.  I'm sure you are all experienced and maybe you have advice?  If 
>you do I would
>apreciate it so much.  The first and best advice get their choice of FTD 
>flowers and/ or
>homemade brownies.
>
>Thank you--Tyler Wellington
>
>
>
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)





------------------------------

Message: 6
Date: Fri, 23 Sep 2005 10:50:34 -0500
From: "Jackie M O'Connor" 
Subject: Re: [Histonet] Let's Play solve this problem....
To: Timothy Macatee 
Cc: Histonetters ,
	histonet-bounces@lists.utsouthwestern.edu
Message-ID:
	

	
Content-Type: text/plain; charset="us-ascii"

Tim -   Are there any prizes for playing?

For IHC it would automatically set off incomplete fixation alarms.  How 
big are your tissues? What kind of tissue are we talking about?
If your tissues are not completely fixed in formalin (or whatever you fix 
in) they are getting primarily fixed in alcohol.
My second instinct would be contamination of your deparaffinization xylene 
(or clearing agent) in your staining set up, not allowing the hematoxylin 
to get to the nuclei because there is an invisible coating of paraffin - 
which of course is then completely removed in the last clearing xylenes.
Third - Incomplete processing - smell your block - no really, go ahead - 
smell your block.  Does it smell like any reagent in particular?  You 
shouldn't be able to smell anything if the processing was right.
But, I sense myself rambling . . . . 
Jackie O'






Timothy Macatee 
Sent by: histonet-bounces@lists.utsouthwestern.edu
09/23/2005 09:49 AM

 
        To:     Histonetters 
        cc: 
        Subject:        [Histonet] Let's Play solve this problem....


Histonetters Assemble!

I'm having a recurring problem with my H & E stained slides.  The staining
is weak, especially away from the edges of the tissue.  It looks almost 
like
there is still an opaque paraffin haze to the tissue.  It could be a
processing problem because it occurs more towards the middle of the 
section.
Is something not getting into the tissue far enough?  Is it Fixative (PFA 
or
Formalin), or processing reagents?  Or am I just whistling dixie?

Tim Macatee
NYU Medical Center
Research Histology Core



_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet




------------------------------

Message: 7
Date: Fri, 23 Sep 2005 08:57:14 -0700
From: "Melissa Gonzalez" 
Subject: [Histonet] Re: Antigen retrieval on mouse GFAP
To: 
Cc: Susan.Ferrigon@sanofi-aventis.com
Message-ID:
	
Content-Type: text/plain;	charset="iso-8859-1"

Susan, I have to agree with Geoff on Dako's GFAP. It is fantastic and
doesn't require Ag retrieval. 
It picks up astrocytes beautifully in mouse brain. I would definitely
recommend DAKO if you aren't using it already. 

Melissa


Melissa A. Gonzalez
Histology 
Cell Genesys
500 Forbes Blvd. 
South San Francisco, CA 94080


Message: 25
Date: Fri, 23 Sep 2005 10:15:06 -0400
From: Geoff McAuliffe 
Subject: Re: [Histonet] Antigen retreival on mouse GFAP.
To: Susan.Ferrigon@sanofi-aventis.com
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <43340DEA.2080803@umdnj.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi Susan:

    I don't know why you would need antigen retrieval for GFAP on mouse 
brain. Most people I know use DAKO's rabbit anti-cow antibody, catalogue 
# Z0334. It works very well on both fixed, frozen sections and on 
formalin-fixed wax embedded tissue. With a Vector ABC Elite kit my 
dilution for GFAP primary is 1:2000 or even more dilute, depending on 
how I intensify the DAB. I also get great results with fluorescence. 
Perhaps your trouble is somewere else?

Geoff

Susan.Ferrigon@sanofi-aventis.com wrote:

>Hi
>
>Does anyone have any experience with using trypsin as the method of antigen
>retreival when staining GFAP on mouse brains???  Any advice on
>concentration, temperature and incubation time would be appreciated.
>
>Thanks
>
>Susan.
>
>----------------------------------------------------------
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-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff@umdnj.edu
**********************************************





------------------------------

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Histonet mailing list
Histonet@lists.utsouthwestern.edu
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End of Histonet Digest, Vol 22, Issue 28
****************************************



------------------------------

Message: 8
Date: Fri, 23 Sep 2005 10:02:35 -0600
From: Gayle Callis 
Subject: Re: [Histonet] removing silver from counters, etc.
To: "Hofecker, Jennifer L" ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050923095708.01b7f9c0@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Put a concentrated solution of i.e 10% or more sodium thiosulfate on top of 
the silver spot, could be a cloth or paper towel soaked in this, and check 
the progress.    I recovered a damaged blouse this way years ago.

  I also suggest using tabletop "diapers" or bench protectors (disposable) 
to prevent spills onto surfaces on the future, a simple paper towel works 
too.


At 07:23 AM 9/23/2005, you wrote:
>Happy Friday everyone.
>Can somebody remind me what solutions will remove silver nitrate from 
>cabinets, floors, counters, etc.?
>Bleach just changes these spots to different shades of brown.  I remember 
>using solution from hair perms in the past  but I am sure that there are 
>other reliable ways.
>Thanks for any advice.  Our lab is starting to resemble a dalmation!  You 
>know how silver solution likes to migrate from the coplin jars!
>
>Thoughts and prayers go out to Gulf Coast residents.
>
>Jennifer
>
>
>
>Jennifer Hofecker, HT (ASCP)
>Vanderbilt University Medical Center
>Division of Neuropathology
>(615) 343-0083
>(615) 343-7089 fax
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)





------------------------------

Message: 9
Date: Fri, 23 Sep 2005 10:03:28 -0600
From: Gayle Callis 
Subject: Re: [Histonet] antigen retrevial/ slides
To: Steven Coakley ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050923100253.01b96650@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

What retrieval are you having problems with and what kind of slides do you 
use now?

At 07:41 AM 9/23/2005, you wrote:
>Are there any recommendations for a good adhesive slide to use for 
>IHC/antigen retrevial.  Also method for drying these slides
>
>Steve
>
>
>---------------------------------
>Yahoo! for Good
>  Click here to donate to the Hurricane Katrina relief effort.
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)





------------------------------

Message: 10
Date: Fri, 23 Sep 2005 10:04:47 -0600
From: Gayle Callis 
Subject: "Hypo" is RE: [Histonet] removing silver from counters, etc.
To: "GUTIERREZ, JUAN" ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050923100352.01c36d40@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

"Hypo" is sodium thiosulfate, folks!!

At 08:03 AM 9/23/2005, you wrote:
>I'm not sure about counters, but for skin I've always use potassium
>ferrocyanide followed by Hypo.
>
>Juan C. Gutierrez, HT(ASCP)
>Histology Laboratory Supervisor
>(210)704-2533

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)





------------------------------

Message: 11
Date: Fri, 23 Sep 2005 12:34:28 -0400
From: "Vinnie Della Speranza" 
Subject: [Histonet] Re:sectioning problems
To: , ,
	
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

I completely agree with Gayle that your clearance angle sounds like the
culprit.

However, on the outside chance that you made have changed the brand of
blades you use, the facet angle may be different which would also require
you to adjust the clearance angle to get the tilt just right to avoid
compression or sections lifting from the blade. 

I'm not convinced that the 64 degrees of your paraffin would have caused the
problem you describe.



Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> Gayle Callis  09/23/05 11:41AM >>>
First and foremost, if you are having the ribbon pulling up as chuck with 
block (Point #3) you need to make sure  knife angle setting on the 
microtome is correct.  Check your manufacturers instructions on HOW to set 
a correct knife angle for your particular microtome.  it sound as though 
the clearance angle is incorrect.  I don't think the paraffin is the 
problem, see last comments.

You did not say what tissues you are sectioning animal?  human? mouse? nor 
how the tissue is processed?  If the tissues are dehydrated, cleared and 
infiltrated with paraffin - optimally for your particular tissue? then you 
should experience good sectioning with correct microtome settings.  Beware 
of infiltrating tissues with excessively hot paraffin, 60C is all that we 
allow with all your animal tisues, over that - I think and with long 
infiltration times, you dry out and create some sectioning problems.

64C is pretty hot in my estimation for embedding, with ANY paraffin.  We 
set ours at 60C only.  You have hot and cold surfaces on your embedding 
center to help you out, these are usually preset at factory although we can 
change that with our Sakura embedding center.  Too hot is an enemy, as you 
cook the tissues even more.  Even the kind of blades you use will affect 
how your sectioning goes, and we all have our favorites - be sure to try 
others on the market!!!

Dirrerent brand paraffins will have different sectioning qualities 
depending on what additives they add to make sectioning easier, etc.  Some 
paraffins are sticky, others are harder, some you have more compression, 
but with care and correct knife angle setting, one can usually learn to 
work with ANY paraffin.   These additives will cause the paraffins to look 
different too - opacity, clarity, hardness, ribboning ability, tackiness as 
you have noticed.

Although I like flowers, I favor the brownies!!!





At 11:20 PM 9/22/2005, you wrote:
>I recently have been having problems pulling ribbons from my microtome and 
>was
>wondering if anyone has advice.
>
>1.) I was having problems pulling ribbons from newly embeded blocks, but 
>ones that had
>been embeded previously were very easy to pull ribbons from.  The new ones 
>would simply
>curl on the blade and wouldn't even grab on to the adjascent sections.
>
>2.) I changed the paraffin in the embedding station because I noticed all 
>the temeratures
>were set to 64 C when the melting point for the Surgipath paraffin formula 
>'R' was 56-67C.
>Could this have been a problem?
>
>3.) I switched to an old lot of Fisher embeding medium and now all the 
>section come off, but
>on occasion when the chuck on the mictomome comes back up it seems to (via 
>static
>electricity?) pull the ribbon from the microtome blade and interupt the 
>ribbon.
>
>4.) Also, visually the Fisher paraffin blocks look almost transparent 
>whereas the Surgipath
>blocks seem opaque and white.
>
>I'm relatively new to the histology game and I realize that there is both 
>art and science
>involved, but this is clearly unacceptable behavior from the paraffin, 
>something is surely
>wrong.  I'm sure you are all experienced and maybe you have advice?  If 
>you do I would
>apreciate it so much.  The first and best advice get their choice of FTD 
>flowers and/ or
>homemade brownies.
>
>Thank you--Tyler Wellington
>
>
>
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu 
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)



_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

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End of Histonet Digest, Vol 22, Issue 29
****************************************

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