[Histonet] RE: Histonet Digest, Vol 115, Issue 17
Natalia Zinchenko
natalia_zinchenko <@t> hotmail.com
Tue Jun 18 14:03:54 CDT 2013
Hello Histonet! I'm a long time reader first time poster. Does anyone have
experience processing guinea pig tissues? I have been processing kidney and
heart but it is consistently coming out mushy in the middle. The mouse
tissue comes out fine even when processed on the same run. I had the tissue
grossed in thinner (2.5mm) thinking that perhaps it was too thick but it
didn't seem to help. Also, it has been fixed in 10%NBF for several days.
I was just wondering if anyone else had similar problems with guinea pig?
I appreciate in advance any advice or tips.
Here is the protocol:
Formalin 1hr
70% etOH 1hr
95% 1hr
100% 30min
100% 1hr
100% 1hr
100%1hr
Clearify 1hr
Clearify 1hr
Clearify 1hr
Paraffin 1hr
paraffin 1hr
All under pressure and heat only on the paraffin.
Thanks
Heather
I work with guinea pig soft and decalcified bone specimens for several years.
Here is my protocol:
Fixation in 10% NBF from24 to 48 hours. Switch to 70% ethanol or do the infiltration
70% etOh 1 hr
80% 1 hr
95% 1 hr
95% 1 hr
100% 1 hr
100% 1 hr
100% 1 hr
Xylene 1 hr
Xylene 1 hr
Xylene 1 hr
Wax58 1 hr
Wax58 1 hr
Wax58 1 hr
Wax58 1 hr
> From: histonet-request <@t> lists.utsouthwestern.edu
> Subject: Histonet Digest, Vol 115, Issue 17
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Mon, 17 Jun 2013 10:00:39 -0700
>
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> Today's Topics:
>
> 1. re 2050 microtome (Steven Weston)
> 2. Cutting paraffin sections on a cryostat operated at room
> temperature? Nope. (Harrison, Sandra C.)
> 3. RE: Paraffin processing native sheep ACL (Jack Ratliff)
> 4. Picro Sirius Red Stain (John Shelley)
> 5. Processing Guinea Pig (Heather Marlatt)
> 6. Re: Picro Sirius Red Stain (Grantham, Andrea L - (algranth))
> 7. Cryostat Repair Service in San Diego (dusko trajkovic)
> 8. Re: Picro Sirius Red Stain (Laura Avogaro)
> 9. ACIS CALIBRATION SLIDE SET (Breal, Kari)
> 10. Re: Processing Guinea Pig (Grantham, Andrea L - (algranth))
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 17 Jun 2013 11:10:17 +0000
> From: Steven Weston <Steven.Weston <@t> utas.edu.au>
> Subject: [Histonet] re 2050 microtome
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <7B808A2E6BDDBD4EA4FA6395FF7BED2B5E7E2402 <@t> MBXSBYN2.utas.ad.internal>
> Content-Type: text/plain; CHARSET=US-ASCII
>
> This could be as simple as moving the specimen holder forward. It may be racked right into the microtome and therefore show as stopped.
>
> Try moving the holder forward and see if the stopped light goes off.
>
> Regards
>
>
> steve weston
> lab manager
> Breathe-Well CRE
> UTAS-SOM
>
>
>
>
>
> Message: 9
> Date: Sat, 15 Jun 2013 00:07:51 -0300
> From: "C.D.G." <latecor <@t> montevideo.com.uy>
> Subject: [Histonet] manual setup for Reichert-Jung 2050 needed
> To: histonet <@t> lists.utsouthwestern.edu
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <201306150007510574.0040B781 <@t> smtp.montevideo.com.uy>
> Content-Type: text/plain; charset="ISO-8859-1"
>
> Hi all:
> i received a Reichert-Jung microtome 2050 model. I need instructions for its operation.
> The electronic panel displays "stop" illuminated and I dont know how to continue, as other
> buttons seems not to operate. Any help of people who has worked or know to operate this
> motorized microtome will be appreciated.
> My kind regards,
> Carlos Defeo
> Histotechnologist
>
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Mon, 17 Jun 2013 08:30:06 -0500
> From: "Harrison, Sandra C." <Sandra.Harrison3 <@t> va.gov>
> Subject: [Histonet] Cutting paraffin sections on a cryostat operated
> at room temperature? Nope.
> To: "Johnson, Kevin" <KJohnson <@t> med.miami.edu>,
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <DB425E28065DA14FAA75A282959620AF073BB215 <@t> VHAV23MSGA2.v23.med.va.gov>
> Content-Type: text/plain; charset="us-ascii"
>
> Ever tried turning the handle of the cryostat, when it's at room
> temperature?
> Cryostats are tooled & manufactured to operate at a low temperature.
> Since metal contracts at the low temperature, you'll find that you can't
> operate the microtome at the higher temperature. The handle will barely
> move.
>
> Sandy Harrison, HTL (ASCP)
> Histology Supervisor
> Minneapolis VAHCS
> 612-467-2449
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Johnson,
> Kevin
> Sent: Friday, June 14, 2013 3:09 PM
> To: 'histonet <@t> lists.utsouthwestern.edu'
> Subject: [Histonet] Cutting paraffin sections...on a cryostat?
>
> Hi, all. A bit of an odd question: a colleague knows of someone wanting
> to cut paraffin sections who has a cryostat, but no microtome. Since a
> cryostat's basically a microtome in a freezer chamber, I thought that it
> may be awkward, but theoretically doable once it was brought to room
> temp and dried out thoroughly. However, I wondered if lubricants
> formulated for the cold might become too thin for use at room temp,
> possibly causing damage to moving parts. Any thoughts?
>
> Kevin Johnson
> University of Miami
> Diabetes Research Institute
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 17 Jun 2013 09:54:53 -0400
> From: Jack Ratliff <ratliffjack <@t> hotmail.com>
> Subject: RE: [Histonet] Paraffin processing native sheep ACL
> To: Andrew Prior <a.prior <@t> tissueregenix.com>, Histonet
> <histonet <@t> lists.utsouthwestern.edu>, "lizronan <@t> umich.edu"
> <lizronan <@t> umich.edu>
> Cc: Jack Ratliff <jratliff <@t> ratliffhistology.com>
> Message-ID: <BLU167-W297DF6CCC1765490C0866BAE830 <@t> phx.gbl>
> Content-Type: text/plain; charset="iso-8859-1"
>
> You might also consider using methyl salicylate instead of xylenes. Thanks to the help of Bob Skinner, I have achieved very nice results with native tendon.
> Generally speaking these MS steps will take a little longer, but you can monitor the progress very easily by watching for complete transparency of the tendon. You can then even develop a somewhat standardized protocol if you plan to process this type of tissue in the future. You even have a lot more flexibility with MS than xylenes as prolonged use in xylenes can make the tissue more hardened and brittle.
> Lastly, it is not generally recommended to put MS on the tissue processor, so I process to the final 100% EtOH, perform the MS exchanges by hand, transfer the tissues into a manual wax step to get rid of as much MS as possible and then finish with three (3) automated wax steps on the tissue processor.
> For my wax infiltration I use a 50:50 blend of TissuePrep from Fisher Scientific and EM400 from Leica. I then embed in 100% EM400.
> Best Regards,
> Jack
>
>
> Jack L RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology
>
>
>
> > From: a.prior <@t> tissueregenix.com
> > To: histonet <@t> lists.utsouthwestern.edu
> > Date: Fri, 14 Jun 2013 07:45:47 +0000
> > Subject: Re: [Histonet] Paraffin processing native sheep ACL
> > CC:
> >
> > Hi Liz,
> >
> >
> >
> > I inherited the following protocol for ACL samples. It works quite well, but times probably could be reduced - the optimization is on my to-do list.
> >
> > 70% Alcohol - 1 hour
> >
> > 90% alcohol - 1 hour
> >
> > 100% Alcohol -2 hours
> >
> > 100% alcohol - 3 hours
> >
> > 100% alcohol - 4 hours
> >
> > Xylene - 1.5 hours
> >
> > Xylene - 1.5 hours
> >
> > Xylene - 3 hours
> >
> > Wax - 3 hours
> >
> > Wax - 3 hours
> >
> > Wax - 4 hours
> >
> > I cut the sections at 8um so they hold together better. Takes a while for all the wrinkles to disappear when floating out on water-bath so be patient
> >
> > Hope this helps.
> >
> >
> >
> > Andrew
> >
> >
> > Andrew Prior
> > Histologist
> > Tissue Regenix Group
> > E-mail: a.prior <@t> tissueregenix.com<mailto:a.prior <@t> tissueregenix.com>
> > Website: www.tissueregenix.com<http://www.tissueregenix.com/>
> >
> >
> >
> > ------------------------------
> >
> > Message: 3
> >
> > Date: Wed, 12 Jun 2013 16:59:52 -0400
> >
> > From: Elizabeth Ronan <lizronan <@t> umich.edu<mailto:lizronan <@t> umich.edu>>
> >
> > Subject: [Histonet] Paraffin processing native sheep ACL
> >
> > To: histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>
> >
> > Message-ID: <51B8E148.3020102 <@t> umich.edu<mailto:51B8E148.3020102 <@t> umich.edu>>
> >
> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> >
> >
> >
> > Hello,
> >
> >
> >
> > I need to paraffin process native sheep anterior cruciate ligament (ACL) that has been fixed in 10% neutral buffered formalin for 7 days. I was wondering if anyone with more expertise on this subject could guide me with the best lengths of times in the alcohols, xylenes, and paraffin to fix ACL. I tried a 12 hour program but the sections crumbled in the middle and it appeared that the paraffin had not fully perfused the ligament.
> >
> >
> >
> > I have access to the following program, and can alter the lengths of the steps for as long as desired:
> >
> >
> >
> > Program:
> >
> > 70%
> >
> > 80%
> >
> > 95%
> >
> > 95%
> >
> > 100%
> >
> > 100%
> >
> > Xylene
> >
> > Xylene
> >
> > Paraffin
> >
> > Paraffin
> >
> > Paraffin
> >
> >
> >
> > Any advice is much appreciated.
> >
> > Thanks for your time,
> >
> > Liz
> >
> >
> >
> > The information transmitted is intended only for the person or entity to which it is addressed and may contain confidential and/or privileged material. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by persons or entities other than the intended recipient is prohibited. If you received this in error, please contact the sender and delete the material from any computer. Although we routinely screen for viruses, addressees should check this e-mail and any attachment for viruses. We make no warranty as to absence of viruses in this e-mail or any attachments.
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>
>
> ------------------------------
>
> Message: 4
> Date: Mon, 17 Jun 2013 14:30:03 +0000
> From: John Shelley <jshelley <@t> sanfordburnham.org>
> Subject: [Histonet] Picro Sirius Red Stain
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <C54F513DA7DA7547B37103A4B74BDAE903BE22C1 <@t> CARRERA.ln.burnham.org>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi All,
>
> Hope all are having a great Monday!!!!
>
> I am trying to do a Picro Sirius Red stain and have looked at many protocols that seem to not use a particular reagent that is in a manufacturers kit. I was hoping if someone could enlighten me as to why this reagent would be used and if someone is using it at what strength is the PhosoMolybdic Acid concentration.
>
> Your help will be greatly appreciated!!!!
>
> Kind Regards!
>
> John J Shelley
> Research Specialist, Histology Core Facility
>
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 17 Jun 2013 07:33:21 -0700
> From: Heather Marlatt <hmarlatt26 <@t> gmail.com>
> Subject: [Histonet] Processing Guinea Pig
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <CALaVVk5w3+UYWkLzEMBo9UOYCRdO3YObpHjajisH8rbSYUTyjA <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello Histonet! I'm a long time reader first time poster. Does anyone have
> experience processing guinea pig tissues? I have been processing kidney and
> heart but it is consistently coming out mushy in the middle. The mouse
> tissue comes out fine even when processed on the same run. I had the tissue
> grossed in thinner (2.5mm) thinking that perhaps it was too thick but it
> didn't seem to help. Also, it has been fixed in 10%NBF for several days.
>
> I was just wondering if anyone else had similar problems with guinea pig?
>
> I appreciate in advance any advice or tips.
>
> Here is the protocol:
>
> Formalin 1hr
> 70% etOH 1hr
> 95% 1hr
> 100% 30min
> 100% 1hr
> 100% 1hr
> 100%1hr
> Clearify 1hr
> Clearify 1hr
> Clearify 1hr
> Paraffin 1hr
> paraffin 1hr
>
>
> All under pressure and heat only on the paraffin.
>
> Thanks
> Heather
>
>
> ------------------------------
>
> Message: 6
> Date: Mon, 17 Jun 2013 15:28:30 +0000
> From: "Grantham, Andrea L - (algranth)" <algranth <@t> email.arizona.edu>
> Subject: Re: [Histonet] Picro Sirius Red Stain
> To: John Shelley <jshelley <@t> sanfordburnham.org>
> Cc: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <02DCE952-FD4D-432E-BB62-84AD17A507D6 <@t> email.arizona.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> I do a Picro Sirius Red stain for collagen (got the protocol from Gayle Callis) and don't use Phosphomolybdic acid.
>
> My protocol is simple:
> one hour in PSR
> two rinses in acidified water
> rinse
> you can counterstain but my clients don't want a counterstain so I dehydrate, clear and coverslip.
> Boom, I'm done.
>
> The PSR stain is just Sirius Red F3B 0.5 gms and 500 ml of Sat. Picric Acid. Do not use a dye that is not CI 35780.
> The acidified water is 5ml Glacial Acetic Acid to 1L of DH2O
>
> Under polarized light the PSR stain is gorgeous! I love looking at the orange, yellow, pinks and green colors. Good thing because I do this stain all the time.
>
> Andrea Grantham, HT (ASCP)
> Senior Research Specialist
> University of Arizona
> Cellular and Molecular Medicine
> Histology Service Laboratory
> P.O.Box 245044
> Tucson, AZ 85724
>
> algranth <@t> email.arizona.edu<mailto:algranth <@t> email.arizona.edu>
> Tel: 520.626.4415 Fax: 520.626.2097
>
>
>
>
>
>
>
> ------------------------------
>
> Message: 7
> Date: Mon, 17 Jun 2013 08:35:43 -0700 (PDT)
> From: dusko trajkovic <dunatrsd <@t> sbcglobal.net>
> Subject: [Histonet] Cryostat Repair Service in San Diego
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <1371483343.29781.YahooMailNeo <@t> web181703.mail.ne1.yahoo.com>
> Content-Type: text/plain; charset=us-ascii
>
>
>
> Good Morning,
> Can anyone recomend a good, reliable cryostat repair service in the San diego area?
> thanks
> Dusko
>
> ------------------------------
>
> Message: 8
> Date: Mon, 17 Jun 2013 17:35:44 +0200 (CEST)
> From: "Laura Avogaro" <avogaro <@t> science.unitn.it>
> Subject: Re: [Histonet] Picro Sirius Red Stain
> To: "John Shelley" <jshelley <@t> sanfordburnham.org>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <3388.192.168.178.78.1371483344.squirrel <@t> www.science.unitn.it>
> Content-Type: text/plain;charset=iso-8859-1
>
> Dear John,
> I usually perform Picrosirius Red stain in cardiac tisse sections.
> I use phosphomolibic acid treatment in order to eliminate the cytoplasmic
> staining improving collagen/myoplasm contrast.
>
> Have a look at this paper:
> Picrosirius red staining of cardiac muscle following phosphomolybdic acid
> treatment
> Dolber PC, Spach MS
> Stain Technol. 1987 Jan;62(1):23-6.
>
> Good luck!
>
> Laura
>
>
> > Hi All,
> >
> > Hope all are having a great Monday!!!!
> >
> > I am trying to do a Picro Sirius Red stain and have looked at many
> > protocols that seem to not use a particular reagent that is in a
> > manufacturers kit. I was hoping if someone could enlighten me as to why
> > this reagent would be used and if someone is using it at what strength is
> > the PhosoMolybdic Acid concentration.
> >
> > Your help will be greatly appreciated!!!!
> >
> > Kind Regards!
> >
> > John J Shelley
> > Research Specialist, Histology Core Facility
> >
> > _______________________________________________
> > Histonet mailing list
> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
>
>
> Laura Avogaro
> University of Trento
> Via delle Regole, 101 38123 Mattarello (TN) – Italy
> Tel: +39 0461 283425
>
>
>
>
> ------------------------------
>
> Message: 9
> Date: Mon, 17 Jun 2013 10:40:32 -0500
> From: "Breal, Kari" <Kari.Breal <@t> alexian.net>
> Subject: [Histonet] ACIS CALIBRATION SLIDE SET
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <B6C4AD10178C854D90B74F853D86C1C2012A25D9D7DF <@t> AUSP03VMBX09.apptixhealth.net>
>
> Content-Type: text/plain; charset="us-ascii"
>
> Does anyone have an ACIS Calibration set they are not using and are willing to part with? One of my slides broke and I can't find a replacement anywhere.
>
> Thanks,
> Kari Breal
>
>
> Kari Breal, HT (ASCP)
> Histology Manager
> Alexian Brothers Health System
> ABMC-847-437-5500 ext. 5155
> SAMC-847-843-2000 ext. 6818
> kari.breal <@t> alexian.net<mailto:kari.breal <@t> alexian.net>
>
>
>
>
>
> CONFIDENTIALITY NOTICE:
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> ------------------------------
>
> Message: 10
> Date: Mon, 17 Jun 2013 15:50:09 +0000
> From: "Grantham, Andrea L - (algranth)" <algranth <@t> email.arizona.edu>
> Subject: Re: [Histonet] Processing Guinea Pig
> Cc: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <C459BA6A-CD33-4BA3-964B-2D2DAA5D51E7 <@t> email.arizona.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> Heather,
> Looking at your protocol for processing the kidney and heart tissue I can't figure out why it is mushy especially if you are putting it in the cassettes so thin and it has had a chance to fix well before processing. In fact, looking at your protocol I might think that your tissue might be dried out and need to sit in cold icy water before sectioning.
>
> At any rate, I would rinse well in running water after fixing and skip the formalin on the processor.
>
> I'd start in 70% alcohol, maybe two changes and move on up to 80%, 2- 95%'s and 3-100%'s.
> I'm not familiar with the clearing agent that you use. I use Clear Rite 3 and never have a problem - 2 changes of Clear Rite 3 and 4 paraffins.
>
> I don't know how many cassettes you are processing but make sure there is adequate room for the reagents on the processor to move around the cassettes and that you have a good ratio of the reagents to the tissue and that the reagents are fresh. If you can increase the agitation in some of the dehydration steps it might help.
>
>
> Andrea Grantham, HT (ASCP)
> Senior Research Specialist
> University of Arizona
> Cellular and Molecular Medicine
> Histology Service Laboratory
> P.O.Box 245044
> Tucson, AZ 85724
>
> algranth <@t> email.arizona.edu<mailto:algranth <@t> email.arizona.edu>
> Tel: 520.626.4415 Fax: 520.626.2097
>
>
>
>
>
>
>
> ------------------------------
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> End of Histonet Digest, Vol 115, Issue 17
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