[Histonet] Re: Cell Pellets in agar
Aprill Watanabe
awatanabe <@t> tgen.org
Tue Jan 17 09:24:01 CST 2006
This is the protocol that I use and it works very well.
Preparation of Cell Culture Blocks as TMA Block Donors
1) Passage cell lines 1.5-2 days before intended harvest date to ensure
cells are in log phase of the cell growth curve.
2) Use flasks that are 75-85% confluent.
3) If using adherent cells, detach the cells with trypsin while treating
cells gently.
4) Collect detached cells, centrifuge for 10 mins at 500xg.
5) Wash once in 1X PBS and pellet again. Remove supernatant. Proceed to
step #9.
6) If using suspension cells, pellet 50 ml of cell culture supernatant in
50 ml conical tubes at 500xg for 10 min.
7) Aspirate off supernatant.
8) Wash with 1X PBS and pellet again. Remove supernatant.
9) Suspend cell pellets in 10 ml of 10% Neutral Buffered Formalin
10) Incubate at 4OC for 2-3 hrs (for suspension cells), overnight on shaker
at room temperature for adherent cells.
11) Make up a 0.8-1% agarose solution and store at 60OC.
12) Add 100 ul of agarose to labeled eppendorf tubes and allow to harden.
13) Pellet cells at 500xg for 10 min.
14) Aspirate off supernatant (Try and get as much as possible)
15) Add 1 drop of Eosin to each cell pellet in the 50 ml tubes.
16) Incubate at RT for 5 min.
17) Suspended cell pellet in 300 ml of warm agarose.
a. Keep the agarose in the 60OC chamber.
b. You only get about enough time to pipet them up and down 3-4 times.
c. Use 1 ml tips with the ends cut off so they don¹t get clogged.
d. Be careful while suspending pellets as bubbles form quite easily.
18) Add agar-cell suspension to respective eppendorf with agar plug.
a. Add a 10 ml pipet tip to plug to allow you to pull the plugs out.
19) Allow agar-cell suspension to harden for 15 minutes.
20) Add ~1 ml of 10% Neutral Buffered Formalin to each tube.
21) Incubate O/N at 4OC.
Send for paraffin embedding the following morning using short protocol.
On 1/16/06 11:39 AM, "histonet-request <@t> lists.utsouthwestern.edu"
<histonet-request <@t> lists.utsouthwestern.edu> wrote:
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> Today's Topics:
>
> 1. RE: RE: Eosin too pink (Rittman, Barry R)
> 2. marker of endothelial differentiation in vitro (Annette Ekblond)
> 3. Desperately needing suggestions please! (Kristen Broomall)
> 4. Cell Pellets in Agar (Johns, Laura [CNTUS])
> 5. autofluorescence (Sasa Jovanovic)
> 6. RE: Dr Marshall & the English Language
> (Marshall Terry Dr, Consultant Histopathologist)
> 7. DI Water (Tyler W.)
> 8. RE: DI Water (Hansen, Donna (Ontario))
> 9. RE: Cell Pellets in Agar (Monfils, Paul)
> 10. RE: Desperately needing suggestions please! (Monfils, Paul)
> 11. RE: Cell Pellets in Agar (Helen Fedor)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sun, 15 Jan 2006 12:28:11 -0600
> From: "Rittman, Barry R" <Barry.R.Rittman <@t> uth.tmc.edu>
> Subject: RE: [Histonet] RE: Eosin too pink
> To: "histonet" <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <EA1FDD2A141B7448B4B1AFFFCAC08DE403AB8688 <@t> UTHEVS1.mail.uthouston.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> If I were in your shoes I would try a weaker solution of esoin and if
> necessary prolong the times.
> I am also a believer in timing the stages of staining, as one perons two dips
> equals another persons three dips and so on.
>
> One would think that considering the amount of time that we all have been
> doing H and Es, that there should be a published standard image that we all
> could agree upon. This ain't gonna happen as the amount of staining with
> eosin that is regarded as desirable is generally a matter of personal taste.
> When stainig for our pathologist many moons ago, we stained deeply with eosin,
> when staning for the researcher the eosin was extermely weak. I am not
> convinced that either of them had a good grasp of the finer points of staining
> but they were the customers.
>
> I think that in talking about eosin staining, it should be borne in mind that
> the type of staining with aqueous and with alcoholic eosin solutions can
> differ considerably.
> Here in the states people have generally used alcoholic solutions of eosin.
> This gives a more rapid stain but in my opinion does not give the range of
> hues that can be obtained with aqueous solutions.
> I am used to using a solution of an aqueous solution, a mixture of eosin Y and
> eosin B (4 part to 1 part). Staining with this for 5 minutes and then
> carrying out most of the differentiation in tap water followed by a rapid
> dehydration. This used for all tissue except bone where a much more dilute
> solution for longer times is best.
> I believe that this provides a range of hues to the eosin staining that allows
> significantly better differentiation of structures. Heavy eosin staining tends
> to lose this differntiation.
> The only time that I used to stain more heavily with esoin was for
> photographing sections, and that is not necessary anymore due to the improved
> imaging techniques and manipulations that are easy to carry out.
> I think that as opinions are bound to differ, what we need is to compare what
> individuals regard as a good H and Es.
> How about sending in photos of what you regard as "good H and Es" to the
> Histonet picture site?
> I suggest that there be a limited number of tissues so that we do not end up
> with a logistic nightmare.
> How about liver, small intestine, uterus, skeletal muscle and skin?
> I think that for bone this might be best via the Hard Tissue Communique.
> Just my opinion.
> Barry
>
> ________________________________
>
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Mark Adam Tarango
> Sent: Sat 1/14/2006 8:03 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] RE: Eosin too pink
>
>
>
> So is he complaining that the eosin is too strong or just too pink? It seems
> to me like the more water you have in the alcohol after Eosin or if you use a
> water rinse after eosin it gets pinker....it's more orange when you have a
> higher percentage of alcohol in the alcohol after eosin(just seems that way to
> me anyway). If it's just too strong, dilute it down with alcohol. I hardly
> get any specimens, so I don't like changing the Eosin often, i just keep
> adding alcohol to it when it gets low and crusted around the edges..... still
> seems to work fine....
>
> Mark T.
>
>> Sent: Friday, January 13, 2006 10:42 AM
>> To: histonet <@t> lists.utsouthwestern.edu
>> Subject: [Histonet] Eosin too pink
>>
>> Hi List,
>> We are currently doing our H & E stain by hand and using the
>> Richard-Allan 7211 Hematoxylin and their Eosin Y. Our pathologist is
>> still complaining of slides being TOO PINK. I have cut the eosin step
>> down to just 5 dips from 15 dips, and still too pink. The Eosin is very
>> rich.
>>
>> This is our protocol:
>> Hydrate to water like normal (thru Clear Rite 3, 100% and 95% Flex)
>> Hematoxylin- 2 mins
>> Rinsing in water until the water runs clear
>> Clarifier II- 30 dips
>> running water -30 dips
>> Richard Allan Bluing - 30 dips
>> running water - 30 dips
>> 95% Flex - 10 dips
>> Eosin-Y- 5 quick dips
>> Thru 95%, 100% etc to xylene
>>
>> Any suggestions on a possible method change or a more mild Eosin.
>> Complaint seems to solely "TOO PINK". I have done some experimenting w/
>> cutting the eosin w/ 80% Flex- and it does calm it down a bit but, the
>> Hemo looks more purply then blue, almost periwinkle so not sure if that
>> will be better or not- we still need to have him review the test slides.
>> Any suggestions ?
>>
>> Helayne Parker
>> Histology Section Head
>> Skaggs Community Health Center
>> Branson, Missouri
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Mon, 16 Jan 2006 11:05:28 +0100
> From: Annette Ekblond <DKAEK <@t> coloplast.com>
> Subject: [Histonet] marker of endothelial differentiation in vitro
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <OF388195EB.21AC519B-ONC12570F8.0035B1EB-C12570F8.00376E90 <@t> coloplast.com>
>
> Content-Type: text/plain; charset=US-ASCII
>
>
> Dear subscribers
>
> -does anyone know of an endothelial marker specific to endothelial cells that
> have differentiated into tube-like
> structures (in vitro) - and not proliferating endothelial cultures??
>
> Kind regards
>
> Annette
> CR
> Humlebaek
> Denmark
>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 16 Jan 2006 08:31:32 -0500
> From: "Kristen Broomall" <kbroomal <@t> NEMOURS.ORG>
> Subject: [Histonet] Desperately needing suggestions please!
> To: Histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <6E41111281623B4B8A9AB8F9A7EA34378244C9 <@t> wlmmsx02.nemours.org>
> Content-Type: text/plain; charset=iso-8859-1
>
> Does anyone know what I can use to get pararosaniline out of fabric? I was
> going to try acid alcohol, but haven't tried anything yet out of fear of
> making it worse.
>
> If anyone has suggestions, I and my brand new tan corduroy jack would much
> appreciate it!
>
> Kristen
>
> kbroomal <@t> nemours.org
>
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Mon, 16 Jan 2006 08:59:12 -0500
> From: "Johns, Laura [CNTUS]" <LJohns53 <@t> CNTUS.JNJ.COM>
> Subject: [Histonet] Cell Pellets in Agar
> To: "'histonet <@t> lists.utsouthwestern.edu'"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <70F83FE9F65318468A612768E7043F8903753FE6 <@t> cntusmaexs9.na.jnj.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Does anyone have a protocol for paraffin embedding cell pellets? We've had
> problems with cell pellets staying together after fixation and during
> embedding, and I know that occasionally people use agar or agarose to keep
> the pellets compact. Is it better to use agar or agarose?
>
> Thanks,
>
> Laura M. Johns, Ph.D.
> Centocor, Inc.
> 145 King of Prussia Road (Mail Stop # R-4-2)
> Radnor, PA 19087
> Phone: 610-240-8284 .... Fax: 610-240-8150.... Email:
> ljohns53 <@t> cntus.jnj.com
>
>> Confidentiality Notice: This message is intended for the individual(s) or
>> entity to which it is addressed and may contain information that is
>> privileged, confidential and exempt from disclosure under applicable law.
>> If the reader of this message is not the intended recipient, he/she is
>> hereby notified that any dissemination, distribution or copying of this
>> communication is strictly prohibited. If you have received this
>> communication in error, please notify the sender by telephone and delete
>> the e-mail from your system immediately. Thank you.
>>
>>
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 16 Jan 2006 16:17:24 +0200
> From: "Sasa Jovanovic" <sasa <@t> jovanovic.co.za>
> Subject: [Histonet] autofluorescence
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <000701c61aa7$9c07b810$640aa8c0 <@t> SinisaHome>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi everyone
> Please help! In my project I am using immunohostochemistry to determine
> presence/absence of integrins in rat uterine tissue. I am trying to make it
> work for several months now with both alpha V beta 3 (immunoflorescence) and
> alpha 4 beta 1 integrin (using DAKO ARK kit) and use of monoclonal
> antibodies. My biggest problem so far is staining in the negative control
> (positive and negative control stain exactly the same) and auto florescence.
> I am following the Santa Cruz Protocol for immuno-fluorescence staining of
> frozen tissue. I snap freeze rat uterine tissue in liquid nitrogen, cut 6 um
> thin sections and adhere them on silane pre-treated slides and dry
> overnight...... then I fix them in cold acetone for 10 minutes and proceed
> with staining as follows:
> - wash slides in three changes of PBS (pH9)
> -incubate slides for 5-10 minutes in 0.1 H2O2 in PBS to quench endogenous
> peroxidase activity
> - wash slides in PBS 2x5minutes
> - incubate slides with 10% normal goat blocking serum in PBS for 20 min to
> suppress non - specific binding of Ig G
> - wash in PBS (3x5min)
> - incubate with primary antibody for 60 minutes
> - wash in 3 changes of PBS
> - incubate for 45 minutes with FITC conjugated secondary diluted in PBS with
> 1.5-3% normal blocking serum in dark chamber
> - wash in 3 changes of PBS
> - mount and coverslip directly from PBS with aqueous mounting medium
> Both primary (sc-7312)and secondary (sc-2010) antibody were purchased from
> SCBT
> I hope this is sufficient info to help you find some answers to my problem
> Thank you in advance
> Kind regards
> Sasha
>
>
> ------------------------------
>
> Message: 6
> Date: Mon, 16 Jan 2006 14:27:15 -0000
> From: "Marshall Terry Dr, Consultant Histopathologist"
> <Terry.Marshall <@t> rothgen.nhs.uk>
> Subject: RE: [Histonet] Dr Marshall & the English Language
> To: <mtitford <@t> aol.com>, <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <FE2DB935F8BBB546B8A1BBF3459C5A1F05F54913 <@t> LIL.xRothGen.nhs.uk>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hmm.
> Can't have it too pink means one likes it pink. I did not realise in using
> this idiom that it is not universally understood.
> That's the problem with idioms.
> One does not ordinarily want to "detect eosinophilia". For that, the merest
> whiff of eosin is what is needed, and that is too my eye, hopeless for
> diagnostic work.
> Perhaps Mike means that there is not the nuance of tint if overstained. Of
> course. By overstating the case I was emphasising my preference to having a
> blue and red stain to a blue and itsi bitsi pink-in-places stain:_)
>
> Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path
> Consultant Pathologist
> Rotherham General Hospital
> South Yorkshire
> England
> terry.marshall <@t> rothgen.nhs.uk
>
> -----Original Message-----
> From: mtitford <@t> aol.com [mailto:mtitford <@t> aol.com]
> Sent: 13 January 2006 18:06
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Dr Marshall & the English Language
>
>
> Dr Terry Marshall sayeth about H & E's that "he cannot have it too pink". Does
> he mean that he does not want it too pink, or that he likes it pink?! In any
> event, how can you readily detect eosinophilia when the eosin is too heavy?
>
> Mike Titford
> Pathology
> USA Mobile AL USA
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
> ------------------------------
>
> Message: 7
> Date: Mon, 16 Jan 2006 8:49:49 -0600
> From: Tyler W. <tyler-wellington <@t> northwestern.edu>
> Subject: [Histonet] DI Water
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <20060116144949.AB5D09E83C <@t> merle.it.northwestern.edu>
> Content-Type: text/plain
>
> The DI water tap here in the lab isn't working. Does anyone know if its
> alright to use regular tap
> water in a flotation bath?
>
>
>
>
>
> ------------------------------
>
> Message: 8
> Date: Mon, 16 Jan 2006 08:27:35 -0700
> From: "Hansen, Donna \(Ontario\)" <DonnaHansen <@t> chiwest.com>
> Subject: RE: [Histonet] DI Water
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <91FAC8D14C98974F839CA842BBC67D42136356 <@t> nwont2dc01.ontario-or.catholichealth.n
> et>
>
> Content-Type: text/plain; charset="iso-8859-1"
>
>
>
> ________________________________
>
> From: Hansen, Donna (Ontario)
> Sent: Mon 1/16/2006 8:21 AM
> To: Tyler W.
> Subject: RE: [Histonet] DI Water
>
>
> Good morning.
> We have always used tap water in the water bath. flotation bath, water bath,
> same thing?
>
> ________________________________
>
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Tyler W.
> Sent: Mon 1/16/2006 7:49 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] DI Water
>
>
>
> The DI water tap here in the lab isn't working. Does anyone know if its
> alright to use regular tap
> water in a flotation bath?
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
>
> ------------------------------
>
> Message: 9
> Date: Mon, 16 Jan 2006 10:31:14 -0500
> From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
> Subject: RE: [Histonet] Cell Pellets in Agar
> To: "'histonet <@t> lists.utsouthwestern.edu'"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <09C945920A6B654199F7A58A1D7D1FDE01717643 <@t> lsexch.lsmaster.lifespan.org>
>
> Content-Type: text/plain; charset="iso-8859-1"
>
> You can use a number of soluble, coagulable proteins to bind cell
> pellets together, including agar, agarose, gelatin, albumin, serum, etc. I
> use Mayer's albumin, but the technique is pretty much the same regardless of
> which substance is used. After fixing the cells in suspension, I spin them
> down and remove the fixative. Then wash a couple of times in buffer by
> resuspending and spinning down. After the last wash I resuspend in buffer
> once more, add 2 drops of Mayer's albumin per ml of suspension, mix
> thoroughly by vortexing a few seconds, allow to stand for a couple of
> minutes, then spin down once more and remove the supernatant. I then begin
> dehydration of the pellet in the centrifuge tube, usually beginning with 50%
> ethanol, then 70%, 95%, and absolute ethanol. Pipette the initial alcohol
> gently down the side of the tube to ensure that the pellet is not disturbed.
> Once you are in the stronger alcohols the thin coating of albumin on the
> cells has coagulated, binding them firmly together. At some point in the
> dehydration series the pellet, due to contraction during dehydration,
> usually detaches from the tube intact. If it doesn't actually detach, it can
> be detached either by drawing up alcohol into a pipette and squirting it
> onto the pellet, or if necessary, by the slightest touch of a slender probe.
>
> I then either wrap the pellet in lens paper and place it in a
> cassette on the tissue processor, starting with the final absolute alcohol
> station, or, I finish the processing by hand, in the centrifuge tube. If
> you are going to do the latter, make sure your tube is resistant to the
> clearing agent. Polypropylene tubes work best for me. Polystyrene tubes
> are more transparent, and are fine if you are only going to use alcohol in
> the tube, but many clearing agents dissolve polystyrene.
>
>
>
>> ----------
>> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
>> Johns, Laura [CNTUS]
>> Sent: Monday, January 16, 2006 5:59 AM
>> To: 'histonet <@t> lists.utsouthwestern.edu'
>> Subject: [Histonet] Cell Pellets in Agar
>>
>> Does anyone have a protocol for paraffin embedding cell pellets? We've
>> had
>> problems with cell pellets staying together after fixation and during
>> embedding, and I know that occasionally people use agar or agarose to keep
>> the pellets compact. Is it better to use agar or agarose?
>>
>> Thanks,
>>
>> Laura M. Johns, Ph.D.
>> Centocor, Inc.
>> 145 King of Prussia Road (Mail Stop # R-4-2)
>> Radnor, PA 19087
>> Phone: 610-240-8284 .... Fax: 610-240-8150.... Email:
>> ljohns53 <@t> cntus.jnj.com
>>
>>> Confidentiality Notice: This message is intended for the individual(s)
>> or
>>> entity to which it is addressed and may contain information that is
>>> privileged, confidential and exempt from disclosure under applicable
>> law.
>>> If the reader of this message is not the intended recipient, he/she is
>>> hereby notified that any dissemination, distribution or copying of this
>>> communication is strictly prohibited. If you have received this
>>> communication in error, please notify the sender by telephone and delete
>>> the e-mail from your system immediately. Thank you.
>>>
>>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>
>
>
> ------------------------------
>
> Message: 10
> Date: Mon, 16 Jan 2006 10:57:35 -0500
> From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
> Subject: RE: [Histonet] Desperately needing suggestions please!
> To: "'histonet <@t> lists.utsouthwestern.edu'"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <09C945920A6B654199F7A58A1D7D1FDE01717644 <@t> lsexch.lsmaster.lifespan.org>
>
> Content-Type: text/plain; charset="iso-8859-1"
>
> For basic dyes like pararosaniline or hematoxylin, acid alcohol would
> probably be your best bet. I keep a little bottle of acid alcohol (fairly
> strong - about 4% hydrochloric acid in 70% ethanol) in the cabinet above the
> washer at home, to pretreat such stains. Acid dyes like eosin or fast green
> will usually wash out pretty easily without pretreatment because of the
> basic pH of the tap water and the detergent. But those same basic
> properties will set basic dyes rather than remove them. I just saturate the
> stain with a little acid alcohol, work it in a bit, then blot it firmly
> between paper towels (under and on top of the fabric) (pound on it a bit
> actually), repeating if necessary, and then when I have sufficiently removed
> the stain, drop the garment into the already running washer. Sounds like
> your concern may be a non-washable garment, in which case the saturate and
> blot technique alone will HOPEFULLY be sufficient. In that case I think I
> would finish up with one last saturate and blot using either plain 70%
> alcohol or distilled water, to remove excess acid from the fabric.
>
> I haven't seen a case where acid alcohol had any direct effect on fabric
> color, but you might want to try it on an inconspicuous part of the garment
> first, just to be sure. My wife once got some black grease on a white
> blouse that had little green leaves and pink and blue flowers. I brought the
> blouse in to work and treated it with xylene, which didn't completely remove
> the grease. They I tried chloroform, which quickly removed the grease, and
> the blue flowers.
>
>
>
>
> ------------------------------
>
> Message: 11
> Date: Mon, 16 Jan 2006 10:59:07 -0500
> From: "Helen Fedor" <hfedor <@t> jhmi.edu>
> Subject: RE: [Histonet] Cell Pellets in Agar
> To: <PMonfils <@t> Lifespan.org>,<histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <s3cb7c87.003 <@t> cis27.hosts.jhmi.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> Paul, Here is the protocol that we use in our Lab.
> Helen
>
> Helen L. Fedor B.S.
> Johns Hopkins University
> Pathology Department
> 600 N Wolfe St
> Marburg Room 406
> Baltimore MD 21287
> email: hfedor <@t> jhmi.edu
> Phone: 410 614-1660
> Pager: 410 283-3419
>
> WARNING: E-mail sent over the Internet is not secure. Information sent by
> e-mail may not remain confidential.
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>
>
>
>>>> "Monfils, Paul" <PMonfils <@t> Lifespan.org> 01/16/06 10:31 AM >>>
> You can use a number of soluble, coagulable proteins to bind cell
> pellets together, including agar, agarose, gelatin, albumin, serum, etc. I
> use Mayer's albumin, but the technique is pretty much the same regardless of
> which substance is used. After fixing the cells in suspension, I spin them
> down and remove the fixative. Then wash a couple of times in buffer by
> resuspending and spinning down. After the last wash I resuspend in buffer
> once more, add 2 drops of Mayer's albumin per ml of suspension, mix
> thoroughly by vortexing a few seconds, allow to stand for a couple of
> minutes, then spin down once more and remove the supernatant. I then begin
> dehydration of the pellet in the centrifuge tube, usually beginning with 50%
> ethanol, then 70%, 95%, and absolute ethanol. Pipette the initial alcohol
> gently down the side of the tube to ensure that the pellet is not disturbed.
> Once you are in the stronger alcohols the thin coating of albumin on the
> cells has coagulated, binding them firmly together. At some point in the
> dehydration series the pellet, due to contraction during dehydration,
> usually detaches from the tube intact. If it doesn't actually detach, it can
> be detached either by drawing up alcohol into a pipette and squirting it
> onto the pellet, or if necessary, by the slightest touch of a slender probe.
>
> I then either wrap the pellet in lens paper and place it in a
> cassette on the tissue processor, starting with the final absolute alcohol
> station, or, I finish the processing by hand, in the centrifuge tube. If
> you are going to do the latter, make sure your tube is resistant to the
> clearing agent. Polypropylene tubes work best for me. Polystyrene tubes
> are more transparent, and are fine if you are only going to use alcohol in
> the tube, but many clearing agents dissolve polystyrene.
>
>
>
>> ----------
>> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
>> Johns, Laura [CNTUS]
>> Sent: Monday, January 16, 2006 5:59 AM
>> To: 'histonet <@t> lists.utsouthwestern.edu'
>> Subject: [Histonet] Cell Pellets in Agar
>>
>> Does anyone have a protocol for paraffin embedding cell pellets? We've
>> had
>> problems with cell pellets staying together after fixation and during
>> embedding, and I know that occasionally people use agar or agarose to keep
>> the pellets compact. Is it better to use agar or agarose?
>>
>> Thanks,
>>
>> Laura M. Johns, Ph.D.
>> Centocor, Inc.
>> 145 King of Prussia Road (Mail Stop # R-4-2)
>> Radnor, PA 19087
>> Phone: 610-240-8284 .... Fax: 610-240-8150.... Email:
>> ljohns53 <@t> cntus.jnj.com
>>
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> End of Histonet Digest, Vol 26, Issue 17
> ****************************************
Aprill Watanabe, B.S.
Research Associate
Tissue Microarray Center (TMA)
Translational Genomics Research Institute (TGen)
602-343-8822
awatanabe <@t> tgen.org
www.tgen.org
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