[Histonet] RE: Histonet Digest, Vol 75, Issue 11
John O'Brien
john <@t> imebinc.com
Sun Feb 7 15:41:53 CST 2010
I may have a folder of old cartoon from year past.
J
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Subject: Histonet Digest, Vol 75, Issue 11
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Today's Topics:
1. We use the microwave processor and we love it we have about 5
. Works great fir small tissue not large. The only issue is
when
it breaks it has to be sent to the shop they send you a
loaner
but you are with out one for a few days so you must have a
back
up. Mike ht (Mike)
2. RE: Slide baking before IHC (Patsy Ruegg)
3. RE: GI, Uro, or Derm Path Lab Set Up (Patsy Ruegg)
4. RE: SPAM-LOW: Re: [Histonet] autofluorescence experiment
(Patsy Ruegg)
5. RE: TRAP assay - acid phosphatase (Patsy Ruegg)
6. RE: SPAM-LOW: [Histonet] Re : KP Markers (Patsy Ruegg)
7. RE: SPAM-LOW: [Histonet] Re: Histonet Digest, Vol 74, Issue
34 (Patsy Ruegg)
----------------------------------------------------------------------
Message: 1
Date: Sun, 7 Feb 2010 09:58:47 -0500
From: Mike <michaelbackhus <@t> aol.com>
Subject: [Histonet] We use the microwave processor and we love it we
have about 5 . Works great fir small tissue not large. The
only issue
is when it breaks it has to be sent to the shop they send you
a loaner
but you are with out one for a few days so you must have a
back up.
Mike ht
To: "Histonet <@t> lists.utsouthwestern.edu"
<Histonet <@t> lists.utsouthwestern.edu>
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Sent from my iPhone
------------------------------
Message: 2
Date: Sun, 7 Feb 2010 08:55:22 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] Slide baking before IHC
To: "'Morken, Tim'" <Timothy.Morken <@t> ucsfmedctr.org>, "'Pat Laurie'"
<foreightl <@t> gmail.com>, <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <7B30AD8C22FB469EB4BF70989584020F <@t> prueggihctechlt>
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I concur with Tim, this paper is a good reference for slide drying
temps, as with everything else there is no rule that works for every
antigen, I play it safe and try very hard to air dry draining until
there is no water left on or under the section, and then I bake at temps
not above 60dc for most things, but when it comes to bone and cartilage,
I drain and air dry them and then lay them flat on heat plate at 45 dc
overnight when possible.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Morken,
Tim
Sent: Friday, February 05, 2010 10:24 AM
To: Pat Laurie; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Slide baking before IHC
Here is the reference:
Effect of Slide Drying at 80Deg C on Immunohistochemistry
A.F. Henwood
J Histotechnology, VOl. 28, no. 1, March 2005
If you are an NSH member you can email the NSH office and ask for a pdf
reprint.
The author compares heating the slides at 80C for seven hours to one
hour at 65C. Some antigens were adversely affected, some were not.
Tim Morken
Supervisor, Histology / IPOX
UCSF Medical Center
San Francisco, CA
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Pat
Laurie
Sent: Friday, February 05, 2010 12:13 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Slide baking before IHC
Histonet,
I have heard rather anecdotally that if you bake slides at high temps
(75 degrees to 80 degrees) before IHC for a long period of time (several
hours to days), you may affect antigenicity for some antibodies. Has
there been any study done about this? Also, what if it is a high temp
(around 75 degrees C) for just 20 minutes? Or a low temperature for a
long period of time?
Thanks in advance for your input.
--
Patrick Laurie HT(ASCP)QIHC
plaurie <@t> cellnetix.com _______________________________________________
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Message: 3
Date: Sun, 7 Feb 2010 09:01:51 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] GI, Uro, or Derm Path Lab Set Up
To: "'Nails, Felton'" <flnails <@t> texaschildrens.org>, "'Richard
Cartun'"
<Rcartun <@t> harthosp.org>, <histonet <@t> lists.utsouthwestern.edu>,
"'Timothy
Jay'" <tjay30 <@t> yahoo.com>
Message-ID: <5F4DE71EC794490C9A8681680BAD31D0 <@t> prueggihctechlt>
Content-Type: text/plain; charset="us-ascii"
The one's who flood histonet with questions are better than the ones
running these labs that we never hear from because they don't even know
what histology is much less that histonet exists.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Nails,
Felton
Sent: Thursday, February 04, 2010 1:57 PM
To: 'Richard Cartun'; histonet <@t> lists.utsouthwestern.edu; Timothy Jay
Subject: RE: [Histonet] GI, Uro, or Derm Path Lab Set Up
Especially when these physician hire unqualified people to run these
labs and flood the histonet with their uneducated questions.
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Richard
Cartun
Sent: Thursday, February 04, 2010 2:29 PM
To: histonet <@t> lists.utsouthwestern.edu; Timothy Jay
Subject: Re: [Histonet] GI, Uro, or Derm Path Lab Set Up
We don't need anymore pathology laboratories. What we need is support
of existing laboratories, especially hospital-based labs. GI and GU
physicians are "Cherry-picking" the technical revenue that should be
going to hospital labs. Let's reform health care; make it more
efficient and less expensive. We don't need to be putting more money in
clinicians' pockets.
Richard
Richard W. Cartun, Ph.D.
Director, Histology & Immunopathology
Director, Biospecimen Collection Programs Assistant Director, Anatomic
Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102
(860) 545-1596 Office
(860) 545-2204 Fax
>>> Timothy Jay <tjay30 <@t> yahoo.com> 2/4/2010 1:28 PM >>>
For those needing help putting an in-office path lab together whether
you are GI, Urology, or Derm please send me an email at tjay30 <@t> yahoo.com
or call me at 775-830-1591. I have a consulting business that
specializes in putting these labs together. References provided upon
request.
Timothy Garcia-Jay, MHA
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Message: 4
Date: Sun, 7 Feb 2010 09:15:56 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: SPAM-LOW: Re: [Histonet] autofluorescence experiment
To: "'John Kiernan'" <jkiernan <@t> uwo.ca>,
<Histonet <@t> lists.utsouthwestern.edu>, "'Nicole Collette'"
<collette2 <@t> mail.llnl.gov>
Message-ID: <4F42B29E0417484ABE444429B029E20B <@t> prueggihctechlt>
Content-Type: text/plain; charset="us-ascii"
In my experience the auto fluorescence from formalin fixation is green
seen with the fitc filter so when I am doing IF on formalin fixed
tissues I try to use a different colormetric label such as Texas Red,
that way even though the bone and red cells are autofluoresing it won't
be seen with my TR filter.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John
Kiernan
Sent: Thursday, February 04, 2010 10:26 AM
To: Histonet <@t> lists.utsouthwestern.edu; Nicole Collette
Subject: SPAM-LOW: Re: [Histonet] autofluorescence experiment
I don't know any argument against using both copper sulphate and Sudan
black B, and you have shown that the combination reduces the
autofluorescence of bone. It's interesting that both are applied after
immunofluorescent staining and are reported to cause some reduction of
the desired fluorescence (Schnell et al 1999 J. Histochem. Cytochem.
47:719-730; Baschong et al 2001 J. Histochem. Cytochem. 449:1565-1571).
Earlier chemical methods for suppressing autofluorescence involved
pre-treatment of sections with either a heavy metal compound or a
non-fluorescent dye. 0.2% osmium tetroxide for 5 mins is very effective;
needs hours in slowly running tap water to wash it out before
fluorescent staining. The dye Direct blue 1 (CI 24410), also known as
Chicago sky blue B, Niagara blue 6B and pontamine sky blue, was
recommended by Cowen et al 1985 Histochemistry 82:205-208 and Kutvolgyi
et al 2006 Biotech. Histochem. 81:4-6. Cowen et al used a 0.05%
solution of the dye in PBS with 1% DMSO. I haven't treid it myself.
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: Nicole Collette <collette2 <@t> mail.llnl.gov>
Date: Wednesday, February 3, 2010 19:12
Subject: [Histonet] autofluorescence experiment
To: Histonet <@t> lists.utsouthwestern.edu
> Hello, All,
>
> I am working on doing some IF stains with bone samples (lucky
> me!). I
> am having a difficult time sometimes to assess the antibody
> since the
> autofluorescence gets in the way. I am using undecalcified, FFPE
> sections (late embryo and neonate mouse bones). Without
> treatment, I
> see autofluorescence everywhere, but most frustrating is the red
> blood cells and the mineralized matrix. It took me a while to
> get
> samples that are properly fixed, section well, and stay on the
> slides, so I am not particularly jazzed about messing with the
> fixation protocol. Thus, I conducted an experiment today of
> several
> published and voodoo methods to reduce autofluorescence with
> samples
> that did NOT undergo the IF protocol :
>
> no treatment
> photobleaching, fluorescent light box, up to 2 weeks photobleaching,
> UV crosslinking light, 2 inches from source, 24h 10mM copper sulfate
> in 50mM ammonium acetate, pH 5.0 0.25% (v/v) NH3 in 70% ethanol (in
> water) 0.25% (v/v) NH4OH in 70% ethanol (in water) (since it was
> unclear
> from the published reference what the source of ammonia was, and
> I
> have made this mistake before on some other thing)
> 0.3% (w/v) Sudan Black in 70% ethanol (in water)
>
> I found that the most effective treatment in my hands is Sudan
> Black
> for cell-based autofluorescence, but it did not seem to impact
> the
> autofluorescence from the mineralized matrix at all, while
> copper
> sulfate had significant impact on the autofluorescence in
> mineralized
> bone, but did not quench cell-based autofluorescence as well as
> the
> Sudan Black (it had an even but incomplete impact over the
> entire
> section). I have tried Sudan Black on my slides before, and have
> found that it did not seem to interfere significantly with the
> antibody signal. I modified the Sudan Black protocol to
> eliminate the
> "goopies" and "chunkies" resulting on my slides from previous
> attempts, and am happy with the results- a light, even stain.
>
> My question to all you chemistry folks: Is there some reason why
> copper sulfate treatment followed by washing and subsequent
> Sudan
> Black treatment (and more washing) cannot or should not be used?
> I
> tried them together on my unstained slides, they look pretty
> darn
> fabulous. Just striving for clean data and beautiful pictures.
>
> Thanks again for all your help,
> Sincerely,
>
> Nicole Collette
> Lawrence Livermore National Lab/ UC Berkeley
>
> _______________________________________________
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------------------------------
Message: 5
Date: Sun, 7 Feb 2010 09:24:35 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] TRAP assay - acid phosphatase
To: "'Liz Chlipala'" <liz <@t> premierlab.com>, "'Sherwood, Margaret '"
<MSHERWOOD <@t> PARTNERS.ORG>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <8B857727EA5340B1B93C5F4B9076D568 <@t> prueggihctechlt>
Content-Type: text/plain; charset="us-ascii"
I use TRAP enzyme histochemical assay from Sigma for osteoclasts on
formalin fixed EDTA decaled Bone paraffin sections, like Liz mentioned
as well as undecalcified frozen bone sections cut with a permanent
tungsten carbide blade in the cryostat using the Instrumedics tape
transfer system. My frozen bone sections are also formalin fixed and
sucrose infiltrated before freezing.
I do not fix the frozen sections because they are already fixed in
formalin. One thing I do with that assay that may not be mentioned in
the protocol which is mainly for smears, is that I incubate at 37dc for
at least an hour and sometimes for 4 hours or more on paraffin or frozen
tape sections.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Wednesday, February 03, 2010 3:49 PM
To: Sherwood, Margaret ; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] TRAP assay - acid phosphatase
We have used it on FFPE EDTA decaled mouse bone, with a little bit of
fiddling with the protocol it works pretty good
Liz
________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Sherwood,
Margaret
Sent: Wed 2/3/2010 3:10 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] TRAP assay - acid phosphatase
Has anybody used the TRAP assay kit from Sigma for bone? We want to
use it on mouse tibia for IHC.
If so, 1) can you do it on paraffin and/or frozen sections?
2) if you do it on frozens, do you decalcify first?
3) what type of blade do you use for sectioning on the cryostat?
3) what procedure do you follow after sectioning: do you fix
slides (i.e. acetone, etc.)
Thank you.
Peggy
Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDW 214)
Massachusetts General Hospital
55 Fruit Street
Boston, Massachusetts 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherwood <@t> partners.org
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------------------------------
Message: 6
Date: Sun, 7 Feb 2010 09:48:46 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: SPAM-LOW: [Histonet] Re : KP Markers
To: "'Vanessa Gutierrez'" <vygutz <@t> yahoo.com>,
<Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <4F268DA564C74653A288BEC184389340 <@t> prueggihctechlt>
Content-Type: text/plain; charset="iso-8859-1"
Yea we just got ours but they were on BO for a few months.
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Vanessa
Gutierrez
Sent: Friday, January 29, 2010 7:42 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: [Histonet] Re : KP Markers
KP Marker pens are sold by Mercedes Medical. I use them and I really
like them. The box I ordered is still on back order so you want to order
as soon as possible.
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Message: 7
Date: Sun, 7 Feb 2010 10:01:15 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: SPAM-LOW: [Histonet] Re: Histonet Digest, Vol 74, Issue
34
To: "'Rhonda Henshall-Powell'" <rlhenshall_powell <@t> yahoo.co.uk>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <C88023E09F3941DE95105BA7FF52AA3B <@t> prueggihctechlt>
Content-Type: text/plain; charset="iso-8859-1"
What I do is make a cell block. If you can get the spheroids into
suspension in a test tube, you could fix in formalin, spin them down in
a centrifuge and then take of the supernatant, add warmed histogel and
resuspend in the histogel, spin it down to get your spheroid in the
bottom of the tube, then let the histogel cool so it will harden, you
can then tease the hardened plug out of the tube, wrap it in paper and
process into paraffin, keep track of the bottom where your cells should
be and embed them down, they should be on the surface of your paraffin
tissue block for sectioning.
Another way is if you can see the spheroids, after fixing, move them
with fine forceps, you could fill an embedding mold with warm liquid
histogel, then place the spheroid in the mold, cool the histogel til it
hardens, then pop it out of the mold and wrap in paper and process into
paraffin as the cell block above. We get HISTOGEL from Richard Allan.
Regards,
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rhonda
Henshall-Powell
Sent: Thursday, January 28, 2010 12:09 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: [Histonet] Re: Histonet Digest, Vol 74, Issue 34
Dear Nick,
When I was growing and harvesting 3D cell cultures (spheroids grown in
matrigel) we would remove the culture medium, draw up the gel in a
needle-less syringe and place in to OCT embedding medium (Tissue Tek)
before freezing in Liquid Nitrogen. I would then cut 5-10um sections
and perform IHC or immunofluorescence.
Hope this helps - but it looks like you have a lot of good suggestions
already.
Best Regards,
Rhonda
Rhonda Henshall-Powell, Ph.D.
>
> ------------------------------
>
> Message: 2
> Date: Wed, 27 Jan 2010 10:25:42 -0800 (PST)
> From: Nicholas David Evans <ndevans <@t> stanford.edu>
>
> Dear all,
>
> I was hoping someone might be able to offer me some advice
> on embedding and sectioning cell cultures.
>
> In short we are growing cells which form 3D dome-like structures on
> tissue culture plastic. Does anyone have any experience or advice to
> offer on embedding the cultures in situ before sectioning? I have seen
> various methods in the literature, which often use Epon to embed the
> material followed by sawing away the plastic, but if anyone can offer
> some tips on other possible (easier) ways of doing it, or
> can refer me to some useful literature, I'd be very
> grateful.
>
> I would like to have simple 10um sections at 90 degrees to the
> substrate, which I can use for IHC.
>
> Best wishes
> Nick
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 27 Jan 2010 15:19:36 -0500
> From: Geoff McAuliffe <mcauliff <@t> umdnj.edu>
>
> Hi Nick:
>
> You can use the Epon substitutes such as EmBed 812. Fix, osmicate,
> dehydrate as usual, but omit the proplylene oxide as it
> will react with
> the plastic dish. Epon substitutes will mix with ethanol. I
> used 2:1
> then 1:1 then 1:2 ratios of absolute ethanol to "Epon" with
> catalyst
> added with agitation. Then several changes of pure Epon and
> polymerize.
> Yes, you will have to saw away the plastic, if you try to
> section the
> Epon-plastic combo the two will separate.
> How much easier do you want it to be?
>
> Geoff
> ------------------------------
>
> Message: 5
> Date: Wed, 27 Jan 2010 15:39:05 -0500
> From: "Sherwood, Margaret " <MSHERWOOD <@t> PARTNERS.ORG>
> Subject: RE: [HISTONET] embedding cell cultures
> Nick,
>
> I am assuming that your 3D cells only grow on
> plastic. They make plastic cover
> slips which, if your cells attach to them and grow, might make the
> embedding much easier. You would follow the same method as
> stated, but then you could
> invert the coverslips on a beem capsule and separate the
> coverslip from capsule
> with liquid nitrogen. However, I have never done it
> with plastic coverslips
> (only glass), so not sure if they would easily separate
> from capsule with liquid
> nitrogen. If anyone else has done so, please weigh in.
>
> Peggy
>
> ------------------------------
> Message: 6
> Date: Wed, 27 Jan 2010 15:44:37 -0500
> From: Peggy Bisher <mbisher <@t> Princeton.EDU>
> Subject: Re: [HISTONET] embedding cell cultures
>
> I have done just what you are talking about using Aclar. It is a
> plastic embedding film (purchased from EMS). It works great for
> us.
>
> Margaret E. Bisher
> Electron Microscopy & Histology Core Facility Manager Department of
> Molecular Biology Princeton University
> Moffett Laboratory, Room 113
> Princeton, New Jersey
> Office: (609) 258-7026
> Fax: (609) 258-8468
> mbisher <@t> princeton.edu
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