[Histonet] Re: Cell blocks on small buttons

From:





----- Original Message -----
From: histonet-request@lists.utsouthwestern.edu
Date: Wednesday, May 23, 2007 7:13 am
Subject: Histonet Digest, Vol 42, Issue 33
To: histonet@lists.utsouthwestern.edu

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> Today's Topics:
> 
>   1. RE: Histonet Digest, Vol 42, Issue 32 (Kobler, James)
>   2. Ethanol ready-to-use for LCM (Castillos, Luminita)
>   3. RE: NK cells in mouse tissue and PLP fixation (Gayle Callis)
>   4. RE: -20C acetone fixative (C.M. van der Loos)
>   5. RE: RE: -20C acetone fixative (Edwards, R.E.)
>   6. Cryomold adapters (Martha Ward)
>   7. Cell Blocks on small buttons... (Lott, Robert)
>   8. celloidin-ether/alc (Mary Lou Norman)
>   9. Work hours (Patti Loykasek)
>  10. RE: celloidin-ether/alc (Rittman, Barry R)
>  11. Re: Work hours (Rene J Buesa)
>  12. RE: Work hours (Mike Pence)
> 
> 
> -------------------------------------------------------------------
> ---
> 
> Message: 1
> Date: Wed, 23 May 2007 10:20:34 -0400
> From: "Kobler, James" 
> Subject: [Histonet] RE: Histonet Digest, Vol 42, Issue 32
> To: 
> Message-ID:
> 	<6D1DFC2837CEAE4BBBDED596071430854D07D4@PHSXMB4.partners.org>
> Content-Type: text/plain;	charset="us-ascii"
> 
> Dear histonet contributors,
> 
> I would greatly appreciate any advice about antibodies that work 
> well with
> ferret tissue (and any related processing tips).  We are 
> particularly interested
> in antibodies to markers for fibroblasts, myofibroblasts, 
> endothelial, smooth
> muscle and inflammatory cells, as well as extracellular matrix.  
> Thanks very
> much,
> 
> Jim Kobler, Massachusetts General Hospital 
> 
> 
> 
> 
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> 
> ------------------------------
> 
> Message: 2
> Date: Wed, 23 May 2007 10:28:19 -0400
> From: "Castillos, Luminita" 
> Subject: [Histonet] Ethanol ready-to-use for LCM
> To: 
> Message-ID:
> 	
> Content-Type: text/plain;	charset="US-ASCII"
> 
> 
> Hi, 
> 
> Does any know from where to buy ready-to-use ethanol (100%, 95%=2C 70%)
> molecular biology grade and RNase-free, compatible for Laser Capture
> microdissection staining protocol?. Thank you very much.
> 
> L, 
> 
> 
> ------------------------------
> 
> Message: 3
> Date: Wed, 23 May 2007 08:43:03 -0600
> From: Gayle Callis 
> Subject: RE: [Histonet] NK cells in mouse tissue and PLP fixation
> To: Bruce Abaloz ,
> 	Histonet@lists.utsouthwestern.edu
> Message-ID:
> 	<6.0.0.22.1.20070523083057.01b3a148@gemini.msu.montana.edu>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
> 
> Sonya,
> 
> You can try PLP immersion fixation followed by sucrose 
> cryoprotection, but 
> the publication I read (reference was in a Histonet message 
> yesterday) did 
> a fixation study with PLP and other fixatives.  They had little 
> success 
> with the NK1.1 (P136) antibody following immersion fixation with 
> all 
> fixatives, before snap freezing or after fresh tissue frozen 
> sections were 
> cut.  They had nice staining following PERFUSION (with PLP) of the 
> mouse.  After perfusion, and once the tissues are dissected out, 
> then 
> immersion into this fixative should ensure complete fixation.  You 
> may want 
> to do final fixation in PLP  overnight, some do it for 5 - 6 hours 
> longer 
> here, then do the 30% sucrose cryoprotection at 4C. Just remove 
> the tissue 
> from fixative and go to the sucrose solution as Bruce suggested.  
> Small 
> fixed tissues often float on top of the sucrose solution.  A 
> general rule 
> of thumb the that cryoprotection is done, tissues sink to the 
> bottom of the 
> container.
> 
> Some people use a sucrose gradient, often seen in the literature, 
> but we 
> have never found that necessary.  We blot off the excess sucrose 
> solution 
> before embedding in OCT.
> 
> Hopefully you have access to a perfusion methods IF your immersion 
> fails.
> Gayle Callis HTL, HT, MT(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717
> 
> 
>  At 06:12 PM 5/22/2007, you wrote:
> >I just snap frooze the fresh tissue in isopentane on dry ice - 
> this has
> >worked with all the other markers I've been looking at but I 
> gather NK
> >cells are more tricky.
> >I want to try PLP fixation before I freeze so do I put it 
> straight from
> >PLP to 30% sucrose or do I wash/use increasing amounts of sucrose.
> >Once I've left the tissue overnight in sucrose can I then freeze 
> it in
> >TissueTek on dry ice as normal?
> >
> >Thanks
> >Sonya
> >
> >-----Original Message-----
> >From: Gayle Callis [mailto:gcallis@montana.edu]
> >Sent: 22 May 2007 15:49
> >To: Martin S.
> >Subject: Re: [Histonet] NK cells in mouse tissue and PLP fixation
> >
> >How did you fix in the first place?
> >
> >You need to sucrose cryoprotect after PLP fixation, in 30% sucrose
> >overnight BEFORE snap freezing the tissues.
> >
> >At 04:01 AM 5/22/2007, you wrote:
> >>Has anyone done any staining for NK cells in mouse tissue. We 
> have an
> >>antibody against NK1.1 (PK136) but it doesnt seem to work on
> >>fresh-frozen tissues. I have looked through the literature and some
> >>people seem to have got iot to work on fresh-frozens while 
> others have
> >>used PLP fixation before embedding in Tissue Tek.
> >>
> >>Has anyone had any experience with this Ab?
> >>
> >>Also, if I fix the tissues with PLP before freezing then how 
> should I
> >>treat them before outting them in TissueTek to freeze?
> >>
> >>Thanks
> >>Sonya
> >>
> >>
> >>
> >>_____________________=5F_______________________=5F_
> >>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
> >
> >
> >
> >
> >
> >
> >
> >
> >______________________=5F_______________________=5F
> >Histonet mailing list
> >Histonet@lists.utsouthwestern.edu
> >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >--
> >BRUCE ABALOZ
> >HISTOLOGIST PH:61383446282
> >DEPT. of ZOOLOGY EMAIL: brucea@unimelb.edu.au
> >THE UNIVERSITY of MELBOURNE. FAX:61383447909
> >VICTORIA.  AUSTRALIA 3010
> >         Nobody Can Make You Feel Inferior Without YOUR 
> Permission - 
> > Eleanor Roosevelt
> >                     WHETHER YOU THINK YOU CAN-OR WHETHER YOU 
> THINK YOU 
> > CAN'T-YOU'RE RIGHT!!
> >                                              Be reasonable..=2E 
> Demand the 
> > impossible.....
> >                           <')))>><             <')))>>< 
> > <')))>><             <')))>><
> >             P Please consider the environment before printing 
> this e-mail.
> >
> >______________________=5F_______________________=5F
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> >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> ------------------------------
> 
> Message: 4
> Date: Wed, 23 May 2007 16:48:47 +0200
> From: "C.M. van der Loos" 
> Subject: [Histonet] RE: -20C acetone fixative
> To: histonet@lists.utsouthwestern.edu
> Message-ID: <37e62d383380.38338037e62d@amc.uva.nl>
> Content-Type: text/plain; charset="us-ascii"
> 
> 
>   Long  time ago they told me it is a milder fixative at -20C 
> than at RT
>   ???
> 
>   Chris van der Loos, PhD
>   Dept. of Pathology
>   Academic Medical Center M2-230
>   Meibergdreef 9
>   NL-1105 AZ Amsterdam
>   The Netherlands
> 
> 
>   -----Original Message-----
>   From: histonet-bounces@lists.utsouthwestern.edu
>   [[1]mailto:histonet-bounces@lists.utsouthwestern.edu=5D   On  
> Behalf  Of
>   Charles,
>   Roger
>   Sent: Tuesday, May 22, 2007 12:29 PM
>   To: histonet@lists.utsouthwestern.edu
>   Subject: [Histonet] -20C acetone fixative
>   Does any know or remember why acetone, when used as a fixative, is
>   always used "ice cold" or in our case at -20C?
>   Roger Charles
>   Microbiologist
>   Pennsylvania Veterinary Laboratory
>   2305 N Cameron St
>   Harrisburg, PA 17110
>   717-787-8808
> 
> References
> 
>   1. javascript:main.compose('new', 't=histonet-
> bounces@lists.utsouthwestern.edu')
> 
> ------------------------------
> 
> Message: 5
> Date: Wed, 23 May 2007 16:15:14 +0100
> From: "Edwards, R.E." 
> Subject: RE: [Histonet] RE: -20C acetone fixative
> To: "C.M. van der Loos" ,
> 	
> Message-ID:
> 	
> Content-Type: text/plain;	charset="US-ASCII"
> 
> And me, in  particular  for  enzyme histochemistry... 
> 
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of 
> C.M. van
> der Loos
> Sent: 23 May 2007 15:49
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] RE: -20C acetone fixative
> 
> 
>   Long  time ago they told me it is a milder fixative at -20C 
> than at
> RT
>   ???
> 
>   Chris van der Loos, PhD
>   Dept. of Pathology
>   Academic Medical Center M2-230
>   Meibergdreef 9
>   NL-1105 AZ Amsterdam
>   The Netherlands
> 
> 
>   -----Original Message-----
>   From: histonet-bounces@lists.utsouthwestern.edu
>   [[1]mailto:histonet-bounces@lists.utsouthwestern.edu=5D   On  Behalf
> Of
>   Charles,
>   Roger
>   Sent: Tuesday, May 22, 2007 12:29 PM
>   To: histonet@lists.utsouthwestern.edu
>   Subject: [Histonet] -20C acetone fixative
>   Does any know or remember why acetone, when used as a fixative, is
>   always used "ice cold" or in our case at -20C?
>   Roger Charles
>   Microbiologist
>   Pennsylvania Veterinary Laboratory
>   2305 N Cameron St
>   Harrisburg, PA 17110
>   717-787-8808
> 
> References
> 
>   1. javascript:main.compose('new',
> 't=histonet-bounces@lists.utsouthwestern.edu')
> _______________________=5F_______________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> ------------------------------
> 
> Message: 6
> Date: Wed, 23 May 2007 11:02:18 -0400
> From: "Martha Ward" 
> Subject: [Histonet] Cryomold adapters
> To: 
> Message-ID:
> 	<61135F0455D33347B5AAE209B903A3041B1C9EBD@EXCHVS2.medctr.ad=2Ewfubmc.edu>
> 	
> Content-Type: text/plain;	charset="us-ascii"
> 
> I am looking for cryomold adapters and I hope someone can help.  The
> ones we are using have a round center and are a white, hard 
> plastic.  I
> can find adapters that have a square center but that isn't what we 
> want.I have tried the usual vendors but was hoping someone could 
> help me.
> Thanks in advance for  your help.
> 
> Martha Ward, MT (ASCP) QIHC
> Assistant Manager, Molecular Diagnostics Lab
> Wake Forest University Baptist Medical Center
> Medical Center Blvd.
> Winston-Salem, NC 27157
> 336-716-2756
> mward@wfubmc.edu
> 
> 
> 
> ------------------------------
> 
> Message: 7
> Date: Wed, 23 May 2007 10:23:14 -0500
> From: "Lott, Robert"  Subject: [Histonet] Cell Blocks on small buttons...
> To: 
> Message-ID:
> 	<673832E27C45FC4D97EF758FFC777C27020E03DC@CPRTEVS01.triadhospitals.net>
> 	
> Content-Type: text/plain;	charset="us-ascii"
> 
> Hi Everyone,
> 
> We are interested in hearing about different procedures for recovering
> small cytology aspirates (any fluid really!) ... and making a cell 
> blockfrom them using some type of coagulative process/fluid/etc. 
> ... so that
> virtually all of the cells are retained!
> 
> 
> 
> There may be some commercial products available ... would like to hear
> about those as well.!!
> 
> 
> 
> Thanks!!!!
> 
> 
> 
> Robert
> 
> 
> 
> Robert L. Lott, HTL(ASCP)
> 
> Manager, Anatomic Pathology
> 
> LabFirst / Trinity Medical Center - formerly
> 
> Montclair Baptist Medical Center
> 
> 800 Montclair Road
> 
> Birmingham, AL   35213
> 
> 205-592-5388  phone
> 
> 205-592-5646  fax
> 
> robert.lott@triadhospitals.com
> 
> 
> 
> 
> 
> 
> ------------------------------
> 
> Message: 8
> Date: Wed, 23 May 2007 11:26:13 -0400
> From: Mary Lou Norman 
> Subject: [Histonet] celloidin-ether/alc
> To: histonet@lists.utsouthwestern.edu
> Message-ID:
> 	<6.2.1.2.2.20070523111029.04899930@postoffice9.mail.cornell.edu>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
> 
> Hello Histonet,
> 
> Is there any other choice besides the cellodin-ether/alcohol 
> coating? My 
> parloidin is taking 'forever' to dissolve -- I have read a week 
> but I could 
> have used it a week ago.
> 
> Thanks for your help.
> Mary Lou Norman
> 
> 
> 
> 
> ------------------------------
> 
> Message: 9
> Date: Wed, 23 May 2007 08:42:22 -0700
> From: Patti Loykasek 
> Subject: [Histonet] Work hours
> To: histonet 
> Message-ID: 
> Content-Type: text/plain; charset="US-ASCII"
> 
> I was wondering if anyone in histoland has experienced moving from a
> schedule of  standard 8 hour day/5 days a week to a 10 hour day/4 
> days a
> week with techs having a day off during the week. If so, what are 
> the pros
> and cons of this type of schedule. I'm interested in feedback from 
> bothbench techs and supervisor/managers. We are in the initial 
> stages of
> contemplating this and would appreciate info from anyone that has 
> tried it.
> Thank you. 
> 
> 
> Patti Loykasek BS, HTL, QIHC
> PhenoPath Laboratories
> Seattle, WA
> 
> 
> 
> -------------------------------------------------------------------
> ------
> This e-mail message, including any attachments, is for the sole 
> use of
> the intended recipients and may contain privileged information. 
> Any 
> unauthorized review, use, disclosure or distribution is 
> prohibited. If 
> you are not the intended recipient, please contact the sender by e-
> mail 
> and destroy all copies of the original message, or you may call 
> PhenoPath 
> Laboratories, Seattle, WA U.S.A. at (206) 374-9000.
> 
> 
> 
> 
> ------------------------------
> 
> Message: 10
> Date: Wed, 23 May 2007 11:37:45 -0500
> From: "Rittman, Barry R" 
> Subject: RE: [Histonet] celloidin-ether/alc
> To: "Mary Lou Norman" ,
> 	
> Message-ID:
> 	
> Content-Type: text/plain;	charset="iso-8859-1"
> 
> Mary Lou
> Will dissolve much more rapidly if soak the strips  first in 
> absolute ethanol for a day o so. The add the ether.
> Barry
> 
> _______________________=5F________
> 
> From: histonet-bounces@lists.utsouthwestern.edu on behalf of Mary 
> Lou Norman
> Sent: Wed 5/23/2007 10:26 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] celloidin-ether/alc
> 
> 
> 
> Hello Histonet,
> 
> Is there any other choice besides the cellodin-ether/alcohol 
> coating? My
> parloidin is taking 'forever' to dissolve -- I have read a week 
> but I could
> have used it a week ago.
> 
> Thanks for your help.
> Mary Lou Norman
> 
> 
> _______________________=5F_______________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> 
> ------------------------------
> 
> Message: 11
> Date: Wed, 23 May 2007 09:52:11 -0700 (PDT)
> From: Rene J Buesa 
> Subject: Re: [Histonet] Work hours
> To: Patti Loykasek ,	histonet
> 	
> Message-ID: <990345.12651.qm@web61216.mail.yahoo.com=3E
> Content-Type: text/plain; charset=iso-8859-1
> 
> I have tried it.
>  Cons: If yu don't have a flow of specimens in 4 days equivalent 
> to the flow on 5 days, the personnel will be idling some time each 
> day. If specimens or workload is available everybody will have 
> work to do.
>  Pros: it is fantastic having 3 days off weekly. Everybody likes 
> that!  Ren� J.
> 
> Patti Loykasek  wrote:
>  I was wondering if anyone in histoland has experienced moving 
> from a
> schedule of standard 8 hour day/5 days a week to a 10 hour day/4 
> days a
> week with techs having a day off during the week. If so, what are 
> the pros
> and cons of this type of schedule. I'm interested in feedback from 
> bothbench techs and supervisor/managers. We are in the initial 
> stages of
> contemplating this and would appreciate info from anyone that has 
> tried it.
> Thank you. 
> 
> 
> Patti Loykasek BS, HTL, QIHC
> PhenoPath Laboratories
> Seattle, WA
> 
> 
> 
> -------------------------------------------------------------------
> ------
> This e-mail message, including any attachments, is for the sole 
> use of
> the intended recipients and may contain privileged information. 
> Any 
> unauthorized review, use, disclosure or distribution is 
> prohibited. If 
> you are not the intended recipient, please contact the sender by e-
> mail 
> and destroy all copies of the original message, or you may call 
> PhenoPath 
> Laboratories, Seattle, WA U.S.A. at (206) 374-9000.
> 
> 
> _______________________=5F_______________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
>       
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> ------------------------------
> 
> Message: 12
> Date: Wed, 23 May 2007 11:58:01 -0500
> From: "Mike Pence" 
> Subject: RE: [Histonet] Work hours
> To: "Rene J Buesa" ,	"Patti Loykasek"
> 	,	"histonet" 
> Message-ID: <661949901A768E4F9CC16D8AF8F2838CA1C5E3@IS-E2K3.grhs.net>
> Content-Type: text/plain;	charset="iso-8859-1"
> 
> How do you decide who gets which days off! Are they floating days?
> 
> Mike
> 
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-
> bounces@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
> Sent: Wednesday, May 23, 2007 11:52 AM
> To: Patti Loykasek; histonet
> Subject: Re: [Histonet] Work hours
> 
> 
> I have tried it.
>  Cons: If yu don't have a flow of specimens in 4 days equivalent 
> to the flow on 5 days, the personnel will be idling some time each 
> day. If specimens or workload is available everybody will have 
> work to do.
>  Pros: it is fantastic having 3 days off weekly. Everybody likes 
> that!  Ren� J.
> 
> Patti Loykasek  wrote:
>  I was wondering if anyone in histoland has experienced moving 
> from a schedule of standard 8 hour day/5 days a week to a 10 hour 
> day/4 days a week with techs having a day off during the week. If 
> so, what are the pros and cons of this type of schedule. I'm 
> interested in feedback from both bench techs and 
> supervisor/managers. We are in the initial stages of contemplating 
> this and would appreciate info from anyone that has tried it. 
> Thank you. 
> 
> 
> Patti Loykasek BS, HTL, QIHC
> PhenoPath Laboratories
> Seattle, WA
> 
> 
> 
> -------------------------------------------------------------------
> ------
> This e-mail message, including any attachments, is for the sole 
> use of the intended recipients and may contain privileged 
> information. Any 
> unauthorized review, use, disclosure or distribution is 
> prohibited. If 
> you are not the intended recipient, please contact the sender by e-
> mail 
> and destroy all copies of the original message, or you may call 
> PhenoPath 
> Laboratories, Seattle, WA U.S.A. at (206) 374-9000.
> 
> 
> _______________________=5F_______________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
>       
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> 
> 
> 
> ------------------------------
> 
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> 
> End of Histonet Digest, Vol 42, Issue 33
> ****************************************
> Robert,
     I asked someone in our cytology section about your inquiry.  What they do is spin down the specinen in a centrifuge, decant the liquid, add about four drops of "HistoGel" (specimen processing gel from Richard-Allen Scientific, 12 vials - 10 ml per vial, Reorder number HG-4000-012), respin the specimen and collect the gel button, put it into a tissue specimen bag before placing it into the cassette.  I hope this will work for you.

Garret

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