[Histonet] Re: Mouse monoclonal antibody for p-c-Jun (the rest of the message)

From:"Donna L Brooks"

Hello again Dr. WL Huang,

I am not sure why all of my message did not appear.  So, here's the rest.
I suggested you do a titration method to check for the best concentrations of your antibodies to see which combination of concentrations yielded the best signal by settting up an experiment changing only the primary concentration, establish that perimeter and then further experimentation changing only the secondary concentration to establish that perimeter.    I suggest starting with say 1:250, then 1:500, 1:1000 and so on with each and see what happens.  
Also, look at www.vectorlabs.com.  They have the type of "rat adsorbed" products I described.  Jackson Immunologicals does as well.
Hope this helps and I was clear.

Donna Brooks HT(ASCP)
Histotechnologist Specialist
University of Louisville
Department of Anatomical Sciences and Neurobiology

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Today's Topics:

   1. RE: pHing of eosin (Gary Gill)
   2. Muscle biopsies (Vivian.King@CLS.ab.ca)
   3. Re: Histonet Digest, Vol 9, Issue 27 (Donna L Brooks)
   4. RE: Muscle biopsies (Mitchell (Jean))
   5. Re: Muscle biopsies (Mildred Fail)
   6. silver question (Bartlett, Jeanine)
   7. Re: Muscle biopsies (Mary North)
   8. Cutting fresh "squishy" rat brain (Kim Merriam)
   9. T blue for mucin veronal acetate buffer (Kathleen Cormier)
  10. Re: Cutting fresh "squishy" rat brain (Gayle Callis)


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

Message: 1
Date: Tue, 17 Aug 2004 09:49:43 -0500
From: Gary Gill 
Subject: RE: [Histonet] pHing of eosin
To: histonet@lists.utsouthwestern.edu
Message-ID: <0B11620AEE5EFB4FA7A7558339972A8307049A@HALIBUT.dcla.com>
Content-Type: text/plain;	charset="iso-8859-1"

Re dye content variation in biological dyes certified by the Biological
Stain Commission, eosin must contain at least 90% dye to be certified.  The
threshold used to be 80% until about 1996 or so.  Dye content is printed on
the labels of all certified dyes -- a notable exception being hematoxylin,
as it is not a dye per se.  The amount of dye that should be weighed out to
produce the same total dye content (TDC) solution will vary, therefore.

For example, to prepare 1 liter of 0.5% TDC eosin solution (raw dye content
= 90% [0.9]), dissolve 5.55 gm eosin in 1 liter solvent (i.e., 5 gm  0.9 =
5.55 gm).  If the raw dye content were 95%, one would weigh out 5.26 gm
(i.e., 5 gm  0.95 = 5.26 gm).  A difference of 0.29 gm will not make any
visually appreciable difference.

If one were using dyes with lower certification dye content thresholds, the
differences between a corrected and uncorrected amount of dye could be
visibly appreciable (e.g., light green SF yellowish = 65%, orange G = 80%).

Gary Gill



-----Original Message-----
From: Barry R Rittman [mailto:Barry.R.Rittman@uth.tmc.edu] 
Sent: Monday, August 16, 2004 6:37 AM
To: histonet@lists.utsouthwestern.edu
Subject: FW: [Histonet] pHing of eosin


I believe that care should  be used in universally adding  specific amounts
of acetic acid to acidify eosin solutions that differ. 

	I asume that different batches of dye have different dye contents so
that the 5% stain that one user prepares is a little different from that
used by others. Each lab should prepare their eosin with this is mind.
	I usually add a low percentage of acetic acid to the eosin until a
faint opalescence occurs. I have been told that this is the point at which
the dye acid just starts to precipitate. 
	For our eosin solutions this involves 5  drops or so of 2% acetic
acid per 100 ml of 5% eosin.
	Barry

		-----Original Message----- 
		From: histonet-bounces@lists.utsouthwestern.edu on behalf of
Tony Henwood 
		Sent: Sun 8/15/2004 6:27 PM 
		To: 'Daryl Mikita'; histonet@lists.utsouthwestern.edu 
		Cc: 
		Subject: RE: [Histonet] pHing of eosin
		
		

		We use 3% acetic acid
		
		
		Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
		Laboratory Manager
		The Children's Hospital at Westmead,
		Locked Bag 4001, Westmead, 2145, AUSTRALIA.
		Tel: 612 9845 3306
		Fax: 612 9845 3318
		
		-----Original Message-----
		From: Daryl Mikita [mailto:dmikita@wmcnet.org]
		Sent: Saturday, 14 August 2004 5:44 AM
		To: histonet@lists.utsouthwestern.edu
		Subject: [Histonet] pHing of eosin
		
		
		Hello,
		
		We are looking at pHing our eosin to the correct pH, instead
of just using
		it straight from the bottle.  What should we use to adjust
the pH?  Should
		it be 1N sodium hydroxide or  1N hydrochloric acid? 
		
		Thanks
		Daryl
		
		
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------------------------------

Message: 2
Date: Tue, 17 Aug 2004 09:45:19 -0600
From: Vivian.King@CLS.ab.ca
Subject: [Histonet] Muscle biopsies
To: histonet@lists.utsouthwestern.edu
Message-ID: <30C050525B881C4AAFF41E6D16543E681927CF@mail3.cls.ab.ca>
Content-Type: text/plain;	charset="iso-8859-1"

Does anyone out there get muscle biopsies sent to them from far away? (ie: a
few hours transport time)
How are they sent to you? (Fresh? or Frozen in proper orientation?)
I am wondering if anyone has any idea how long a muscle bx can be held on
ice before the diagnostic value of the tissue is compromised. Any
information would be greatly appreciated.

Vivian King 
Tech II - Neuropathology 
FMC 
Calgary, Alberta 
403-944-1576 
vivian.king@cls.ab.ca 

 


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

Message: 3
Date: Tue, 17 Aug 2004 11:50:59 -0400
From: "Donna L Brooks" 
Subject: [Histonet] Re: Histonet Digest, Vol 9, Issue 27
To: 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Hello Dr. WL Huang,

In regards to the mouse monoclonal antibody for p-c-Jun:
Be sure to check if your secondary antibody is "rat adsorbed".  I
suspect that you may be experiencing a cross reactivity due to the
species (rat/mouse)  being so closely related.  I have also done quite a
bit of immunoflourescent and immunoperoxidase staining of rodent neural
tissue from spinal cord injured rodents, and had this very same problem.
  Also, I would suggest checking the Ig groups.  For example, IgG versus
IgA.  Did you use a protein blocking solution?  If not you may want to
try that as well. Be sure to watch the concentration of your antibodies
and blocking serums/protein solutions.  If the concentrations are too
high (say 1:250 [high] vs  1:1000[lower]) you will get a high
background.  I suggest doing a titration method to see which
concentrations give th



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

Message: 4
Date: Tue, 17 Aug 2004 11:22:39 -0500
From: "Mitchell \(Jean\)" 
Subject: RE: [Histonet] Muscle biopsies
To: ,	
Message-ID:
	
	
Content-Type: text/plain;	charset="us-ascii"

There is an article in December 1998 Journal of Histotechnology "Delayed
Processing of Muscle Biopsy Specimens:  Does it Really Compromise Enzyme
Histochemistry?" The paper addresses extended delay (up to 48 hours) in
processing muscle biopsies. I can fax a copy if you would like.

When muscle biopsies are transported to us, ideally we like to receive
the tissue on regular ice (not dry ice) within 3 hours. At times it has
taken up to 6 hours to receive samples. The staining & diagnostic value
of the muscle tissue has remained intact in all cases. With extended
transport time usually PAS staining is the most compromised due to
glycogan leakage. But one can always note the difference in the section
quality of a muscle biopsy that was frozen & sectioned on site vs a
biopsy that was transported to us for processing.

We tend to see more problems with muscle samples when they are frozen on
site then transported to us on dry ice. Much more artifact with that
method - but they are still diagnostic.

Jean Mitchell, BS, HT (ASCP)
University of Wisconsin Hospital & Clinics
Department of Neurology
Madison, WI

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of
Vivian.King@CLS.ab.ca
Sent: Tuesday, August 17, 2004 10:45 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Muscle biopsies

Does anyone out there get muscle biopsies sent to them from far away?
(ie: a few hours transport time) How are they sent to you? (Fresh? or
Frozen in proper orientation?) I am wondering if anyone has any idea how
long a muscle bx can be held on ice before the diagnostic value of the
tissue is compromised. Any information would be greatly appreciated.

Vivian King
Tech II - Neuropathology
FMC
Calgary, Alberta
403-944-1576
vivian.king@cls.ab.ca 

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

Message: 5
Date: Tue, 17 Aug 2004 12:19:44 -0400
From: "Mildred Fail" 
Subject: Re: [Histonet] Muscle biopsies
To: , 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Vivian,
     We have outside clients wrap the  fresh muscle in saline dampened gauze put it in a biohazard bag then place that bag in another containing ice. Most of the time the biopsies are sent by cab or by one of  their security personnel.  But they are sometimes shipped overnight on ice. We did run some tests on Autopsy muscle and saw only  minimal loss of enzyme histochemistry after 72 hours of refrigerator storage.  But we prefer  than  none go  longer than 48 before snap freezing, and specify that the muscles must be received within 24 hours.
Rena Fail

>>>  08/17/04 11:45AM >>>
Does anyone out there get muscle biopsies sent to them from far away? (ie: a
few hours transport time)
How are they sent to you? (Fresh? or Frozen in proper orientation?)
I am wondering if anyone has any idea how long a muscle bx can be held on
ice before the diagnostic value of the tissue is compromised. Any
information would be greatly appreciated.

Vivian King 
Tech II - Neuropathology 
FMC 
Calgary, Alberta 
403-944-1576 
vivian.king@cls.ab.ca 

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

Message: 6
Date: Tue, 17 Aug 2004 12:34:41 -0400
From: "Bartlett, Jeanine" 
Subject: [Histonet] silver question
To: 
Message-ID:
	
Content-Type: text/plain;	charset="us-ascii"

All:
 
Has anyone experienced a problem with their Steiner's "fading" after a
period of time?  We had a slide that had been stained with the Steiner
procedure and it was very positive, however when it was reviewed at a
much later date it appeared negative.  Another section was stained and
it was positive.  I know that leaving the sections for a long time in
xylene before they are coverslipped can lead to fading but wouldn't this
be apparent when the slides were originally viewed?  
 
Any suggestions?
 
Thanks!
Jeanine Bartlett, HT(ASCP)
Centers for Disease Control and Prevention
Infectious Disease Pathology Activity
1600 Clifton Road, MS/G-32
Atlanta, GA 30333


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

Message: 7
Date: Tue, 17 Aug 2004 09:35:36 -0700
From: "Mary North" 
Subject: Re: [Histonet] Muscle biopsies
To: Vivian.King@CLS.ab.ca,	histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: text/plain; charset=us-ascii

In our experience, if the specimen has been kept moist (not soaked in
saline) and cold, the enzyme histochemisty stains still yield diagnostic
results even after many hours following surgery.  Some of our fresh
specimens are not received until the day after surgery due to a faraway
location.  These have been refrigerated at the source hospital and then
sent on ice, FedEx overnight.  
Mary North, HT(ASCP), HTL
Neuromuscular Laboratory
Oregon Health & Science University
Portland, OR

>>>  8/17/2004 8:45:19 AM >>>

Does anyone out there get muscle biopsies sent to them from far away?
(ie: a
few hours transport time)
How are they sent to you? (Fresh? or Frozen in proper orientation?)
I am wondering if anyone has any idea how long a muscle bx can be held
on
ice before the diagnostic value of the tissue is compromised. Any
information would be greatly appreciated.

Vivian King 
Tech II - Neuropathology 
FMC 
Calgary, Alberta 
403-944-1576 
vivian.king@cls.ab.ca 


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

Message: 8
Date: Tue, 17 Aug 2004 09:38:45 -0700 (PDT)
From: Kim Merriam 
Subject: [Histonet] Cutting fresh "squishy" rat brain
To: Histonet 
Message-ID: <20040817163845.22190.qmail@web52505.mail.yahoo.com>
Content-Type: text/plain; charset=us-ascii

Hello everyone,
 
I need to cut sections of fresh rat brain, in certain stereotactic coordinates and then embed them in OCT.  Does anyone have any tips as to how to do this without squashing the brain?  We are trying to cut coronal sections of the brain to precisely expose a particular region, and we are using a fresh disposable microtome blade to do the cuts, but the brain is still very squishy when fresh and is very difficult to cut.   We are doing LCM on these cryosections, so any type of fixation is out of the question.
 
Kim Merriam
Novartis
Cambridge, MA


Kim Merriam
Novartis
Cambridge, MA
		
---------------------------------
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Yahoo! Mail is new and improved - Check it out!

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

Message: 9
Date: Tue, 17 Aug 2004 12:47:58 -0400
From: Kathleen Cormier 
Subject: [Histonet] T blue for mucin veronal acetate buffer
To: histonet@pathology.swmed.edu
Message-ID: <5.2.1.1.2.20040817123802.00af5b40@hesiod>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Hello All!

I am looking for a supplier for the veronal acetate buffer needed in the T 
blue for mucin (Theory and Practice ver 1980 pg 169-70) Since the buffer 
uses sodium barbiturate (alas! a controlled substance!) it would be much 
easier to get this buffer pre made. Any one know of anyone who sells this 
buffer or kit? Is there a similar stain that does not have sodium 
barbiturate? Is this a lost cause? This researcher is looking at gel 
membranes with glycosaminoglycans chains, and would like the T blue to 
stain the sulfated  and  nonsulfated mucopolysaccharides...

Thanks!

Kathy




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

Message: 10
Date: Tue, 17 Aug 2004 10:53:23 -0600
From: Gayle Callis 
Subject: Re: [Histonet] Cutting fresh "squishy" rat brain
To: Kim Merriam ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20040817104743.01b1d888@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

What about holding brain in one of the rat brain matrices holders.  It 
allows holding the brain for precise slices, coronal 
orientation.  www.Myneurolab.com has these, for coronal, sagittal, and 
different age and size of rats.  You can cut as thin as 1 mm

At 10:38 AM 8/17/2004, you wrote:
>Hello everyone,
>
>I need to cut sections of fresh rat brain, in certain stereotactic 
>coordinates and then embed them in OCT.  Does anyone have any tips as to 
>how to do this without squashing the brain?  We are trying to cut coronal 
>sections of the brain to precisely expose a particular region, and we are 
>using a fresh disposable microtome blade to do the cuts, but the brain is 
>still very squishy when fresh and is very difficult to cut.   We are doing 
>LCM on these cryosections, so any type of fixation is out of the question.
>
>Kim Merriam
>Novartis
>Cambridge, MA
>
>
>Kim Merriam
>Novartis
>Cambridge, MA
>
>---------------------------------
>Do you Yahoo!?
>Yahoo! Mail is new and improved - Check it out!
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)





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

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End of Histonet Digest, Vol 9, Issue 28
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