[Histonet] RE: Histonet Digest, Vol 25, Issue 5
Gus Mondragon
gmondragon <@t> gsopath.com
Tue Dec 6 02:29:46 CST 2005
A positive and a negative control is required by CLIA in all AFB stains (Leprosy and TB). A known positive control validates your staining procedure; a negative control eliminates the presence of artifact in your sections Gus Mondragon gmondragon <@t> gsopath.com
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
Sent: Saturday, December 03, 2005 1:09 PM
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Subject: Histonet Digest, Vol 25, Issue 5
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Today's Topics:
1. Re: RE: Paraffin sections (Maria Mejia)
2. Extracellular matrix proteins fixation. Details
(Diego J. Rodr?guez Gil)
3. RE: Specail stains Negative controls (Smith, Jeffery D. (HSC))
4. Re: Specail stains Negative controls (Bryan Hewlett)
5. RE: Goat polymer (pruegg <@t> ihctech.net)
6. Knife holder (arunams)
7. RE: Question: Inventory (Favara, Cynthia (NIH/NIAID))
8. AW: [Histonet] Question: Inventory (Gudrun Lang)
----------------------------------------------------------------------
Message: 1
Date: Fri, 02 Dec 2005 10:16:05 -0800
From: Maria Mejia <maria <@t> ski.org>
Subject: Re: [Histonet] RE: Paraffin sections
To: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>
Cc: histonet <@t> lists.utsouthwestern.edu, "C.M. van der Loos"
<c.m.vanderloos <@t> amc.uva.nl>, BMolinari <@t> heart.thi.tmc.edu
Message-ID: <43908F65.6010808 <@t> ski.org>
Content-Type: text/plain; charset=us-ascii; format=flowed
This type of experimenting would be interesting to check out especially
with
lower percentages of BSA. Andrea, I would agree that this overnight storage step probably is antigen dependent (Chris, that's what I meant by too much) and it would require a whole gamut testing of antibodies to see which
respond
positively and which would not...if one is inclined to do so.
Yours
Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185
Andrea T. Hooper wrote:
> Very interesting Chris! I have found sometimes this works and
> sometimes it doesn't and you get complete loss of staining -
> frustrating when one was just trying to save time to begin with!! I
> imagine it's antigen dependent but I have not had time to check.
>
> In my opinion Betsy, if you can avoid it (because you are just doing
> it to save time instead of setting up a workshop) then avoid it ... it
> simply isn't worth the risk IMHO.
>
>
>
> At 9:52 AM +0100 12/2/05, C.M. van der Loos wrote:
>
>> Betsy,
>>
>> For my NSH wet-workshops I tested the option of going up to endogenous
>> PO blocking, HIER and then overnight storage at 4C in PBS with 1% BSA.
>> Next day we started at the protein blocking step and then the rest.
>> Staining was as good as running the whole procedure in one day.
>>
>> Hope this info helps.
>>
>> Chris van der Loos, PhD
>> Dept. of Pathology
>> Academic Medical Center M2-230
>> Meibergdreef 9
>> NL-1105 AZ Amsterdam
>> The Netherlands
>
>
------------------------------
Message: 2
Date: Fri, 02 Dec 2005 13:55:04 -0500
From: "Diego J. Rodr?guez Gil" <Diego.RodriguezGil <@t> yale.edu>
Subject: [Histonet] Extracellular matrix proteins fixation. Details
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1133549704.43909888f401e <@t> webmail.med.yale.edu>
Content-Type: text/plain; charset=ISO-8859-1
Hi:
Thanks a lot to all of you who answer!. Since some of you asked, I am sending some more details.
I am working on mice (CD-1), and the molecules I am trying to stain are named Wnt (Wingless-Int). Antibodies are from R&D systems (most of them are goats), and there are no previous works reporting the use of these antibodies. I am only making the assumption that they work, based on the datasheet the company provides. I could see some very faint positive stain for some of them, but I am sure I losing some because of fixation. Besides that, I know they are expressed based on RT-PCR and in situ hybridization experiments.
Brief protocol is:
Perfusion with 4% PFA, and then overnight deep in 4% PFA at 4 C. Transfer to PBS, then 30% sucrose til they sink, freeze on OCT and then section on cryostat at -18C.
The antigen retrieval protocols that I tried were steaming (2, 5 and 10 min with sodium citrate) or pepsin in HCl. None of them gave me any better stainings.
Staining protocol: Wash in TBS and then add 2% BSA in TBS-T, for 30 min. Primary antibody (on 2% BSA) Over night .
Wash 3 x TBS-T.
Add secondary antibody on 2% BSA for an hour.
Wash 2 x TBS-T.
Wash TBS and mount with Crystal Mount.
Thanks again!
Best regards,
Diego.
--
Diego J. Rodríguez Gil
Postdoctoral Associate
Department of Neurosurgery
Yale University School of Medicine
PO Box 208082
333 Cedar St, FMB-430
New Haven CT, 06520-8082
U.S.A.
------------------------------
Message: 3
Date: Fri, 2 Dec 2005 13:23:19 -0600
From: "Smith, Jeffery D. \(HSC\)" <Jeffery-Smith <@t> ouhsc.edu>
Subject: RE: [Histonet] Specail stains Negative controls
To: "Grant, Debra, R" <drgrant <@t> cmh.edu>,
<Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<EA80A7E0A540C44A8A339A3561E35C60063E262B <@t> PERCHERON.hsc.net.ou.edu>
Content-Type: text/plain; charset="iso-8859-1"
Debby,
Routine special stains only require a positive control as you are looking to see if the stain worked or not. Specimen is either positive or negative. No need for negative control. IHC uses both pos. and neg. controls because you are looking for varying intensities of staining as well as pos. or neg.
Best of Luck,
Jeff
________________________________
Hi Histonetters,
Does anyone run negative control slides with their GMS, AFB, and Gram special stains, and if so why?
Kind Regards,
Debby R. Grant HT(ASCP)
Histology Coordinator
The Children's Mercy Hospital & Clinics
2401 Gillham Rd.
Kansas City, Missouri 64108
lab (816)234-3827
fax (816) 802-1492
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------------------------------
Message: 4
Date: Fri, 2 Dec 2005 15:23:28 -0500
From: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>
Subject: Re: [Histonet] Specail stains Negative controls
To: "Smith, Jeffery D. (HSC)" <Jeffery-Smith <@t> ouhsc.edu>, "Grant,
Debra, R" <drgrant <@t> cmh.edu>, <Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <002001c5f77e$786f0990$6400a8c0 <@t> mainbox>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
Debby and Jeff,
Negative control slides are useful for GMS, Gram and AFB in order to
eliminate false positives due to contamination from the reagents, washing,
carry-over etc.
Regards,
Bryan
----- Original Message -----
From: "Smith, Jeffery D. (HSC)" <Jeffery-Smith <@t> ouhsc.edu>
To: "Grant, Debra, R" <drgrant <@t> cmh.edu>; <Histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, December 02, 2005 2:23 PM
Subject: RE: [Histonet] Specail stains Negative controls
Debby,
Routine special stains only require a positive control as you are looking
to see if the stain worked or not. Specimen is either positive or negative.
No need for negative control. IHC uses both pos. and neg. controls because
you are looking for varying intensities of staining as well as pos. or neg.
Best of Luck,
Jeff
________________________________
Hi Histonetters,
Does anyone run negative control slides with their GMS, AFB, and Gram special stains, and if so why?
Kind Regards,
Debby R. Grant HT(ASCP)
Histology Coordinator
The Children's Mercy Hospital & Clinics
2401 Gillham Rd.
Kansas City, Missouri 64108
lab (816)234-3827
fax (816) 802-1492
_______________________________________________
Histonet mailing list
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------------------------------
Message: 5
Date: Fri, 2 Dec 2005 13:39:43 -0700
From: pruegg <@t> ihctech.net
Subject: RE: [Histonet] Goat polymer
To: "'Orr, Rebecca'" <ROrr <@t> enh.org>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200512022039.jB2KdkXX005415 <@t> pro12.abac.com>
Content-Type: text/plain; charset="us-ascii"
Becky,
Invitrogen is the company who makes the goat labeled polymer. As for blocking, I use serum free protein block before the primary and then before the rab. Anti-goat secondary and then again before the rabbit labeled polymer. If I still encounter non-specific staining I put some serum (from the host of the secondary, about 10%) in with the protein block. This method is very sensitive and you can bring up lots of non-specific BG so be careful to optimize your dilutions and times for the antibody and for the detection reagents. Patsy
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Orr, Rebecca
Sent: Friday, December 02, 2005 6:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Goat polymer
Hello everyone,
Just recently there was a posting on how to run a rabbit polymer with goat antibodies...or something to that nature, using an anti rabbit secondary and then the Dako polymer...could that person send me an email please? I had a quick question about blocking...
Also, does anyone have the name of the company that makes a goat polymer? I know there's a company out there...I've seen the ads but can't locate it...
Many thanks and have a great weekend!
Becky
Becky Orr CLA,HT(ASCP)
IHC Lead
Evanston Northwestern Healthcare
847-570-2771
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------------------------------
Message: 6
Date: Fri, 02 Dec 2005 14:04:38 -0800 (PST)
From: arunams <arunams <@t> interchange.ubc.ca>
Subject: [Histonet] Knife holder
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<5641228.1133561078869.JavaMail.myubc2 <@t> portal9.itservices.ubc.ca>
Content-Type: text/plain; charset=us-ascii
Hi All
Does eny one know where to find a Tungston Carbide Knive holder for a Sorvall JB-4 Microtome. I really need one urgently. Thanks for your help Aruna Somasiri
------------------------------
Message: 7
Date: Fri, 2 Dec 2005 17:35:26 -0500
From: "Favara, Cynthia \(NIH/NIAID\)" <cfavara <@t> niaid.nih.gov>
Subject: RE: [Histonet] Question: Inventory
To: "Luis Chiriboga" <Luis.Chiriboga <@t> med.nyu.edu>, "Histonet"
<Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<A985B7D45D355D4EB363288E717394375B8090 <@t> NIHCESMLBX5.nih.gov>
Content-Type: text/plain; charset="us-ascii"
I do not do high volume consistently but have some weeks where I am doing 200-300 stains per week. I do not register my reagents as the come into the lab. c
Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317
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-----Original Message-----
From: Luis Chiriboga [mailto:Luis.Chiriboga <@t> med.nyu.edu]
Sent: Thursday, December 01, 2005 11:14 AM
To: Histonet
Subject: [Histonet] Question: Inventory
Hi All
For those of you who are in high volume labs using ventana stainers, do you register all your reagents when they arrive? or do you register as you use? Thanks in advance Luis _______________________________________________
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------------------------------
Message: 8
Date: Sat, 3 Dec 2005 08:52:40 +0100
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] Question: Inventory
To: "Histonetliste \(Histonetliste\)"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<mailman.0.1133632800.18983.histonet <@t> lists.utsouthwestern.edu>
Content-Type: text/plain; charset="us-ascii"
We register the bulk-reagents and prep-kits in the Ventana System just before use. The antibodies are registered, when they arrive, to have them on the list.
Gudrun Lang
Hi All
For those of you who are in high volume labs using ventana stainers, do you register all your reagents when they arrive? or do you register as you use? Thanks in advance Luis _______________________________________________
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