[Histonet] Re: Osteoid staining

From:Damien



Hi Jim,



Does your study require Osteoid measurements in the transverse plane (you
did not mention this)? If so, obviously you will need to use ground sections
(30-40 microns).

If your samples have not been processed, I would recommend doing an
osteochrome bulk stain (the Villanueva method) stain and take your
measurements using fluorescence. (Osteoid will be a vivid orange). Even if
you've already processed, there are still options for using thick sections.
Assuming your polishing technique is consistent (section thickness), you
won't have any analysis problems. It is perfectly acceptable to use thick
sections for osteoid measurement.


-Damien Laudier

On Wed, Sep 17, 2008 at 1:01 PM,
wrote:

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>
>   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.
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> or distribution of this e-mail or use of information contained herein is
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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  <
> http://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:
>        <
> EA1FDD2A141B7448B4B1AFFFCAC08DE40C048465@UTHEVS1.mail.uthouston.edu>
> 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.
>
> http://windowslive.com/connect/post/jamiethomson.spaces.live.com-Blog-cns!550F681DAD532637!5295.entry?ocid=TXT_TAGLM_WL_domore_092008
>
> ------------------------------
>
> 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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