[Histonet] RE: Histonet Digest, Vol 58, Issue 20

From:"Versfeld, BG, Mr "



can someone please provide me with a recipe for hirschprung's disease. thanx.

________________________________

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Sent: Wed 2008/09/17 07:06 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 58, Issue 20



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Today's Topics:

   1. Looking for H&E schedule for Varistain 24-4 (Randall Carpenter)
   2. Re: (no subject) (Geoff McAuliffe)
   3. (no subject) (anita dudley)
   4. Re: Amyloid (Maxim_71@mail.ru)
   5. RE: Amyloid (Mark A Burton)
   6. NSH class (Amber McKenzie)
   7. Permanent histo job in Austin, TX (Kyla Nemitz)
   8. Question on staining (Herrick, James L.)
   9. stain (Paul Verden)
  10. RE: Question on staining (Rittman, Barry R)
  11. job opening (anita dudley)
  12. Re: stain (Ben Spirto)
  13. RE: Question on staining (Jack Ratliff )


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

Message: 1
Date: Tue, 16 Sep 2008 10:45:39 -0700 (GMT-07:00)
From: Randall Carpenter 
Subject: [Histonet] Looking for H&E schedule for Varistain 24-4
To: Histonet  
Message-ID:
        <16648622.1221587139951.JavaMail.root@elwamui-royal.atl.sa.earthlink.net>
       
Content-Type: text/plain; charset=UTF-8

Greetings Histonet,

It's been a while since I was here and now I'm back.  I'm now Twin Cities
Histology and it's a busy business right now.  I just purchased a used Varistain
24-4 and was wondering if anyone had a good schedule for the Harris Hematoxylin
(regressive).  I've been doing the AFP method by hand for the last year or so,
but now I'm up against it and had to buy the machine.  Any help would be appreciated.
Thanks.

Randy Carpenter
Twin Cities Histology



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

Message: 2
Date: Tue, 16 Sep 2008 14:17:48 -0400
From: Geoff McAuliffe 
Subject: Re: [Histonet] (no subject)
To: "TOJO (Torben Seested Johansen)" 
Cc: "histonet@lists.utsouthwestern.edu"
        
Message-ID: <48CFF84C.2060502@umdnj.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Dear Tojo:

    Your problem could be due to 3 things. 1. Waiting too long to fix
the tissue. 2. Freezing too slowly. 3. Interpreting the lack of staining
of hepatocyte glycogen as an artifact.

1. You are waiting 5 minutes after death to fix the tissue. There is
absolutely NO reason to spend 5 min pumping 100 ml of PBS through the
circ. system. You will never wash out all of the blood and there is no
need to. AND when you finally get to fixative, the flow of fixative is
too low. Here is the solution to your problem.

Use a 16 g needle in the L. vent.
Pump 10-15 ml of room temperature PBS into the rat in 15-20 seconds.
Pump 300 ml of room temperatue fix in 5 minutes. The fix is 4%
paraformaldehyde for light microscopy. For transmission EM the fix is 2
or 3 or  4% paraformaldehyde with 1% glutaraldehyde. Buffer can be
either phosphate or cacodylate. The advantage of cac. buffer is that you
can add some calcium salts to stabilize membranes for EM (2milleMole)
without percipitation.Yes, you can add Ca salts to phos. buffer but it
will precipitate instantly (look up the solubility of CaPhosphate, or
should I say the insolubility). Of course, cacodylate has arsenic in it
so don't lick your fingers.
Remove liver and cut into appropriate sized pieces. For light microscopy
fix as long as possible, formalin reacts with tissue very slowly.
Glutaraldehyde fixed much faster, an hour or two is plenty.
Cryoprotect with sucrose.
2. Tissue must be frozen VERY QUICKLY with 2-methylbutane (isopentane)
cooled with dry ice or liquid nitrogen otherwise you will have large
holes due to ice crystal formation.
3. The stain you are using, Tol.Blue will not stain glycogen so you may
have empty-looking areas because of this. Use Periodic acid Schiff for
glycogen but NOT after glutaraldehyde fixation.

Geoff


TOJO (Torben Seested Johansen) wrote:
> Hi,
> I do not seem to be able to achieve acceptble morphology of perfused rat liver. I seems as if some of the cytosol of the hepatocytes are "washed away" as seen in toluidine blue stained cryo-sections (see image18.jpg). I am to use the tissue cryo-sections in immunohistochemistry and transmission electron microscopy.
>
> I my attempts to get an acceptable/good morphology I have tried fixation buffers consisting of 2, 4, 6 or 8 % paraformaldehyde (w/wo 0.1% glutaraldehyde).
>
> My fixing procedure is as follows (all at room temperature);
>
> a rat (~250g) is anasthestized and a perfusion needle is inserted into the left ventricle. The atrium is cut and PBS is flushed thru the rat at 20ml/min for 5min. The rat is thereafter fixated at 10ml/min for 10 min with fixation buffer (I have so far tried 2, 4, 6 or 8 % paraformaldehyde (with and with out 0.1% glutaraldehyde) in 0.1M cacodylatebuffer (pH 7.4) all with same unacceptable result (see image18.jpg). The liver is cut into smaller pieces (~2*2*2mm) post fixed for 1h in the respective fixative, transferred into 2.3M sucrose for minimum 1h  (preferably over night) and frozen in liquid nitrogen.
>
> Any idea why teh cytosol of my hepatocytes seems to be "gone" (hydropic degeneration?) ?
>
> I have searched the net and it seems as if theres a 1000's of different ways of performing rat liver perfusion fixation. Some use cold buffers and others also perfuse with sucrose solution (either in combination with the fixative or with sucrose alone post-fixation)
>
> what can I do to try and optimise my perfusion ?
>
> ______________________________________
>
> Torben Seested Johansen
> Post Doc
> Exploratory ADME, Biopharmaceuticals
>
> Novo Nordisk A/S
> Novo Nordisk Park
> E9.S.22
> DK-2760 Mlv
> Denmark
> +45 44 43 14 84 (direct)
> +45 44 66 39 39 (fax)
> tojo@novonordisk.com
> www.novonordisk.com
>
> This e-mail (including any attachments) is intended for the addressee(s) stated above only and may contain confidential information protected by law. You are hereby notified that any unauthorized reading, disclosure, copying or distribution of this e-mail or use of information contained herein is strictly prohibited and may violate rights to proprietary information. If you are not an intended recipient, please return this e-mail to the sender and delete it immediately hereafter. Thank you.
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>
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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
mcauliff@umdnj.edu
**********************************************






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

Message: 3
Date: Tue, 16 Sep 2008 13:52:42 -0500
From: anita dudley 
Subject: [Histonet] (no subject)
To: "histonet@pathology.swmed.edu" 
Message-ID: 
Content-Type: text/plain; charset="iso-8859-1"

job in fairhope alabama, beautiful area along the bay. dematologist office looking for someone to do the mohs surgery.  contact anita, providence hosp.  mobile alabama,  251-633-1422.
_________________________________________________________________
See how Windows connects the people, information, and fun that are part of your life.
http://clk.atdmt.com/MRT/go/msnnkwxp1020093175mrt/direct/01/

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

Message: 4
Date: Tue, 16 Sep 2008 23:05:58 +0400
From: Maxim_71@mail.ru
Subject: Re: [Histonet] Amyloid
To: Dorothy.L.Webb@HealthPartners.Com
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <596251904.20080916230558@mail.ru>
Content-Type: text/plain; charset=us-ascii

Dorothy:
We does Highman's method (Bancroft&Stevens, 1977 p.164)
Both 8 and 10 microns give good results.
Maxim Peshkov
Russia,
Taganrog.
                     mailto:Maxim_71@mail.ru




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

Message: 5
Date: Tue, 16 Sep 2008 15:31:30 -0400
From: "Mark A Burton" 
Subject: RE: [Histonet] Amyloid
To: "'Webb, Dorothy L'" ,
        
Message-ID: <000c01c91832$d24f65e0$76ee31a0$@edu>
Content-Type: text/plain;       charset="US-ASCII"

Dorothy,

We've done several comparisons like this for amyloid staining. In our
experience, 8um works fine. We did see stronger staining (birefringence) in
some thicker sections with Congo Red but I don't think there would be a
significant improvement in staining (sensitivity?) from 8um to 10um. I
wouldn't cut sections any thinner than 8um but you can still get amyloid
staining at 4um. It would be relatively easy to take your controls and cut
them different thicknesses just to prove the point. Good luck!


Mark A Burton HTL ASCP
Lab Manager & Sr. Histotechnologist
Molecular Aging & Development Lab
Boston University Medical Campus



-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Webb,
Dorothy L
Sent: Tuesday, September 16, 2008 11:17 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Amyloid

We have been cutting our tissue for amyloid staining @ 8 microns.  One
of my pathologists heard that 10 microns is now standard  and to use a
negative and weakly positive control.  Does anyone have any new
information in this area?  Thanks ahead of time!

Dorothy Webb, HT (ASCP)
Histology Technical Supervisor
Regions Hospital, Pathology Department
640 Jackson Street, Saint Paul, MN 55101-2595
Phone: 651-254-2962
Fax: 651-254-2741
Regions Hospital is part of the HealthPartners family of care
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------------------------------

Message: 6
Date: Tue, 16 Sep 2008 14:32:08 -0500
From: "Amber McKenzie" 
Subject: [Histonet] NSH class
To: 
Message-ID:
        <03C921A1EAF7F541B16543F6EC6A4B3701EB6B95@giamail2.Gia.com>
Content-Type: text/plain;       charset="us-ascii"

Hi!  I was wondering if anyone was going to NSH and attending the class
for "Are you Ready for the HT board of Registry Exam"?  by speaker:
Robert L Lott, BS, HTL (ASCP), Trinity Medial Center/LabFirst, Inc.,
Birmingham, AL ?  I am not able to attend NSH this year, and I'm really
interested in the material that Robert will be presenting about. I am
already HT certified, but I want to take the HTL soon and thought this
class would be a good start for study material. I emailed Pebbles at NSH
for the speakers email address to contact him personally, but I never
heard back from her.  If anyone is planning on going to this class,
could you send me a copy of any handouts?



Thanks,

Amber McKenzie, B.S., HT (ASCP)

1405 N. State St., Suite 400

Jackson, MS 39202

(ph) 601-863-0388

(fax) 601-326-3532







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

Message: 7
Date: Tue, 16 Sep 2008 16:24:29 -0400
From: "Kyla Nemitz" 
Subject: [Histonet] Permanent histo job in Austin, TX
To: 
Message-ID:
        <9416D9FA37C1C04FA83D69684E95D2E71E4DDD95@exbk2.maxhealth.com>
Content-Type: text/plain;       charset="us-ascii"

Hello all -



I am looking for an ASCP Histo tech for a permanent position in Austin,
TX. The position is day shift, M-F and pays $55-60K/year. The facility
is also helping with relocation.



If you or anyone you know is currently looking to relocation to Texas,
please contact me.



Also - I specialize in placing histo techs on travel and permanent
positions Nationwide. If you have any questions or are looking for a
position, please feel free to call or email.



Thank you in advance, your help is appreciated!



Kyla Nemitz

TravelMax Medical Professionals - Maxim Healthcare 

* 888.800.1855 or 813.371.5175

7 800.294.1248

www.TravelMaxAllied.com 





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

Message: 8
Date: Tue, 16 Sep 2008 17:11:46 -0500
From: "Herrick, James L." 
Subject: [Histonet] Question on staining
To: 
Message-ID:
        
Content-Type: text/plain;       charset="iso-8859-1"

Hi all,

I am trying to stain for osteoid and bone in pig tibia (need to quantify osteoid and bone volumes). Does anyone have experience with these types of stains in 40-60 m thick, MMA embedded, sections? I have tried a Masson's trichrome and Toluidine blue, but have been unable to get well defined stain differentiation with this thick of sections. I would really appreciate any help I can get. Thanks again.

Jim



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

Message: 9
Date: Wed, 17 Sep 2008 10:29:04 -0500
From: "Paul Verden" 
Subject: [Histonet] stain
To: 
Message-ID:
        
Content-Type: text/plain;       charset="US-ASCII"

I am interested in finding the Alternate Bielschowsky Stain Protocol.
There are problems getting the silver to clear.







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

Message: 10
Date: Wed, 17 Sep 2008 10:38:58 -0500
From: "Rittman, Barry R" 
Subject: RE: [Histonet] Question on staining
To: "Herrick, James L." ,
        
Message-ID:
        
Content-Type: text/plain;       charset="iso-8859-1"

There is a question on the validity of measuring osteoid on thick sections.
The reason is that it is rare in bone to have sections where edges are absolutely vertical throughout the section. You therefore have several planes superimposed and several edges in these planes resulting in a penumbra effect. The greater the angle to the vertical edge of the bone such as edges of trabeculae or Haversian canals, the greater the errors in measuring.
The most accurate measurements are those utilizing sections that are 5 microns or below.
Is there some particular reason that you must use sections this thick?
Barry

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Herrick, James L.
Sent: Tuesday, September 16, 2008 5:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Question on staining

Hi all,

I am trying to stain for osteoid and bone in pig tibia (need to quantify osteoid and bone volumes). Does anyone have experience with these types of stains in 40-60 m thick, MMA embedded, sections? I have tried a Masson's trichrome and Toluidine blue, but have been unable to get well defined stain differentiation with this thick of sections. I would really appreciate any help I can get. Thanks again.

Jim

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



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

Message: 11
Date: Wed, 17 Sep 2008 10:50:47 -0500
From: anita dudley 
Subject: [Histonet] job opening
To: "Histonet@lists.utsouthwestern.edu"
        
Message-ID: 
Content-Type: text/plain; charset="iso-8859-1"

sorry I failed to put anything in the subject line on the previous note.  anita   job is in fairhope ala. mohs lab in need of a histologist.  251-633-1422
_________________________________________________________________
Get more out of the Web. Learn 10 hidden secrets of Windows Live.
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------------------------------

Message: 12
Date: Wed, 17 Sep 2008 10:58:01 -0500
From: "Ben Spirto" 
Subject: Re: [Histonet] stain
To: "Paul Verden" 
Cc: histonet@lists.utsouthwestern.edu
Message-ID:
        <287d127c0809170858g53d73752v6cb4152870407ff1@mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

*Bielschowski Stain*


*20% Silver Nitrate Solution:*
1. Silver Nitrate=20g
2. dWater=100ml


*Ammonia Water:*
1. dWater=100ml
2. Ammonium Hydroxide=8 drops


*Developer:*
1. dWater=100ml
2. Formalin=20ml
3. Citric Acid=0.5g
4. Concentrated Nitric Acid=2 drops
*Carcinogenic*

*5%Sodium Thiosulfate (Hypo)*
1. Sodium Thiosulfate=5g
2.dWater=100ml


Ammonium Silver Solution
1. 20% silver nitrate=100ml
2. Ammonium Hydroxide=10ml


Protocol:

On the stir plate to the 20% silver nitrate solution add 10-15 ml of
ammonium hydroxide, until brown precipitate forms, adding one drop at the
time. Keep adding more of ammonium hydroxide until solution is clear (while
vigorously stirring). Add couple drops of 20 % of silver nitrate until
solution is gold. Slides are placed in the ammonium silver solution in the
dark for 15-20 min. After that slides are placed in ammonia water. To the
ammonium silver solution add  the developing solution (developer) in the
ratio of 8 drops of developer per 100ml of solution while stirring. Slides
are developed 3-5 min

On Wed, Sep 17, 2008 at 10:29 AM, Paul Verden wrote:

> I am interested in finding the Alternate Bielschowsky Stain Protocol.
> There are problems getting the silver to clear.
>
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>


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

Message: 13
Date: Wed, 17 Sep 2008 16:08:21 +0000
From: "Jack Ratliff " 
Subject: RE: [Histonet] Question on staining
To: "Barry.R.Rittman@uth.tmc.edu " ,
        "Herrick.James@mayo.edu " ,
        "histonet@lists.utsouthwestern.edu "
        
Message-ID: 
Content-Type: text/plain; charset="iso-8859-15"

Jim,

I would like to respond to your question regarding the pig tibia. After reading Barry's reply, I too question why you would not want to cut thin (5 micron) sections. It then occurred to me that maybe you are not able to do so because this would require the use of a sledge or polycut microtome.

Given the size of your specimen, I agree that you you will have better and more consistent results with your histomorphometry endpoints if you cut thinner sections using a Reicher-Jung Polycut or a Leica SM2500. Upon completion, you can then deplastify the sections (if you use MMA) and yield excellent staining results by employing a Von Kossa reaction with a MacNeals tetrachrome counterstain and/or a standard Goldners trichrome stain.

If you need further assistance or more information, please feel free to contact me

Jack Ratliff

-----Original Message-----
From: Rittman Barry R 
Sent: Wednesday, September 17, 2008 11:39 AM
To: Herrick.James@mayo.edu; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Question on staining

There is a question on the validity of measuring osteoid on thick sections.
 The reason is that it is rare in bone to have sections where edges are absolutely vertical throughout the section. You therefore have several planes superimposed and several edges in these planes resulting in a penumbra effect. The greater the angle to the vertical edge of the bone such as edges of trabeculae or Haversian canals, the greater the errors in measuring.
 The most accurate measurements are those utilizing sections that are 5 microns or below.
 Is there some particular reason that you must use sections this thick?
 Barry

 -----Original Message-----
 From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Herrick, James L.
 Sent: Tuesday, September 16, 2008 5:12 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Question on staining

 Hi all,

 I am trying to stain for osteoid and bone in pig tibia (need to quantify osteoid and bone volumes). Does anyone have experience with these types of stains in 40-60 m thick, MMA embedded, sections? I have tried a Masson's trichrome and Toluidine blue, but have been unable to get well defined stain differentiation with this thick of sections. I would really appreciate any help I can get. Thanks again.

 Jim

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

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




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

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End of Histonet Digest, Vol 58, Issue 20
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