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>From: "HistoNet Server" <HistoNet@Pathology.swmed.edu>
>To: "HistoNet Server" <HistoNet@Pathology.swmed.edu>
>Subject: Daily Digest
>Date: Thu, 20 May 1999 21:07:40 -0500
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 08:31:16 -0500
>From: jim <jim@proscitech.com.au>
>Subject: RE: Microwave processing/ future lab
>
>Jim -
>If microwave processing is done properly then differences in fixation are
>minute. Note that microwave fixation is used in TEM, so it stands to reason
>that some of the "minute" differences in histo fixation are actually
>improvements on conventional methods. Rapid preparation methods that 
>include
>staining and decalcification may also show minor variations.
>It's not a question of "what is real"; no preservation method gives 
>completely
>
>life-like results. The important question is 'are the differences large 
>enough
>
>to potentially affect interpretation when conventional and microwave 
>fixation
>are employed in the same laboratory'. It seems that the literature (what 
>I've
>seen) suggests that interpretation or diagnosis would not be affected. This 
>is
>
>a question that people experienced with both methods may like to discuss.
>Our online lists some pertinent microwave references and information on a 
>link
>
>from page E3 which is called  . . . . features, applications and some
>references.
>Disclaimer: PST is a supplier of laboratory microwave systems.
>
>Concerning the "future lab". I like the quote "it is difficult to predict,
>especially the future" (forgot who said). It seems that frequently new
>technology does not replace the old but it added as another (expensive)
>option.
>I predict that microwave will be most common fixation in histo labs within
>five
>years and well before Confocals are common in these labs.
>Cheers
>Jim Darley
>ProSciTech                 Microscopy PLUS
>PO Box 111, Thuringowa  QLD  4817  Australia
>Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
>Great microscopy catalogue, 500 Links, MSDS, User Notes
>                       www.proscitech.com.au
>
>On Friday, July 09, 1999 6:39 PM, Jim Hall [SMTP:rmkdhjh@ucl.ac.uk] wrote:
> > Dear colleagues,
> >
> > I am in the final stages of evaluating a microwave tissue processor and 
>am
> > awaiting our pathologists' comments on the morphology.  Does anyone have
> > views on how sections from microwave processed tissues compare to 
>sections
> > produced by enclosed processors morphologically?  I would very much like 
>to
> > hear from anyone who has.
> >
> > Referring to "The future of Histology" I can go back even further when 
>we
> > took delivery in the early 60's of a brand new Slee cryostat complete 
>with
> > rocker microtome and my Pathologist telling me that this was the future 
>and
> > that all sections examined by Pathologists would be frozen sections!!!
> >
> > Best regards,
> >
> > Jim.
> > Jim Hall,
> > MDA Equipment Evaluator,
> > Department of Histopathology,
> > University College London Hospitals,
> > Rockefeller Building,
> > University Street,
> > London, WC1E 6JJ.
> > Tel.No. 0171 209 6042
> > Fax 0171 387 3674
>
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 08:31:58 -0500
>From: jim <jim@proscitech.com.au>
>Subject: RE: Freezing muscle sections/snap freeing technique
>
>Ian - from what I read in your several missives about freezing technique,
>there
>is nothing of substance I would disagree with and that is not because of 
>the
>possible, violent application of a "Lochhaber axe". - Is that gadget used 
>to
>gross specimens?
>
>For those interested in cryo, I would like to comment on a couple of 
>points.
>Hope this will not jeopardise the prospect of some lukewarm amber, which I
>detest, but would imbibe to appease any woad attired high priest of histo.
>
>I think that the danger of exploding propane is over-stated. Yes, I know it
>can
>do so very nicely, but if used within a fumehood no dangerous vapour
>concentration can occur, short of sticking a match right at the container. 
>The
>
>modest size propane cylinder is only used while filling the nitrogen cooled
>"cup".
>I grant that isopentane is more convenient and frequently "good enough" for
>cryo fixation of "bulk" specimen. Previously and now, I am writing about
>possible remedies to poor cryo preservation. Knowing the criteria enables 
>the
>user to make intelligent choices when modifying their methods. As 
>previously
>noted, the larger histo specimen cannot possibly be preserved completely
>crystal free, but the means used to achieve true vitrification would 
>minimize
>crystal interference.
>One important criteria is speed of freezing. Several parameters affect 
>speed,
>including: specimen size, lowest possible temperature of cryo agent and
>conductivity of specimen and agent.
>
>The lowest usable temperature of propane is about 30 degree lower than
>isopentane. That is significant.
>another approach is the use of slushy nitrogen. Liquid nitrogen placed 
>under
>vacuum boils rapidly. Boiling cools a liquid (saucepan with boiling water
>remains at a hundred, despite further heat-input) and since no heat is 
>added
>to
>the liq N2, boiling cools the nitrogen until reaching solidification point 
>-
>it
>looks like crushed ice, but is softer. Slushy nitrogen does not quickly 
>form
>that gas insulating layer which makes liquid nitrogen a useless cryo-agent.
>This material is used in practical cryo work. For instance, the Emitech SEM
>bolt-on preparation system includes a slushy nitrogen system. (Declare
>interest; PST is Emitech agent for Australasia) For those interested in the
>subject, PST's online has an internal link from page K4 (cryo stages) 
>entitled
>
>a "technical brief  on bulk frozen hydrated specimens"
>I appreciate your comment on the slammer method. This of course works well
>because it's for tiny samples only, near liq nitrogen temperature and 
>because
>of pressure and excellent conductive properties of the polished copper 
>anvil.
>(I'll spare you another commercial, but yes PST has a commercial interest).
>The
>interesting facts are, that copper is an excellent conductor at those low
>temperatures, gold and silver are slightly better, but not very practical.
>Stainless Steel conducts heat very poorly at low temperatures. I have used
>thin
>stainless wires to conduct electricity out of a dewar with minimal 
>heat-gain.
>Now Ian, if you really want to go to town buy a diamond anvil. (Sorry, but
>yes,
>I could provide that too) Diamond is the best conductor at those low
>temperatures, it takes an excellent polish and does not scratch easily. I
>think
>that de Beers need a break and a stampede of diamond demanding histos would
>even help the SA economy.
>Ian you really should try a good Ozzie red, beats that lukewarm amber.
>Cheers
>Jim Darley
>ProSciTech                 Microscopy PLUS
>PO Box 111, Thuringowa  QLD  4817  Australia
>Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
>Great microscopy catalogue, 500 Links, MSDS, User Notes
>                       www.proscitech.com.au
>
>On Wednesday, July 07, 1999 8:17 PM, Ian Montgomery
>[SMTP:ian.montgomery@bio.gla.ac.uk] wrote:
> > >Date: Fri, 2 Jul 1999 17:55:47 +1000
> > >From: jim <jim@proscitech.com.au>
> > >Subject: FW: Freezing muscle sections/snap freeing technique
> > >To: "'Tim Fairchild'" <timf@cyllene.uwa.edu.au>,
> > >        HistoNet Server
> > >	 <HistoNet@pathology.swmed.edu>
> > >Mime-Version: 1.0
> > >
> > Jim,
> > 	You got me when I was a wee bit nippy. Family trait, crabbit, bad
> > tempered and quick to reach for the Lochaber axe. I'll buy you a pint 
>first
> > time we meet.
> >
> > >Tim - Isopentane is not the best medium to use!
> > 	Have to disagree, for good muscle histochemistry, the original
> > question, isopentane cooled with liquid nitrogen is essential. Ok, I 
>have
> > used talc coated specimens in the past, but that was only in an 
>emergency.
> >
> > >Isopentane is very sticky and almost unusable when its near liq 
>nitrogen
> > >temperature.
> > 	Isopentane is completely unusable at liquid nitrogen temperature,
> > you have a solid block. Not much use for plunge freezing.
> >
> > Warmed up (with a metal rod) the cooling rate of the specimen is
> > >reduced, because (heat transfer rates of various media aside and liquid
> > >nitrogen is lousy, because it forms an "insulating" gas layer), the 
>colder
> > >the
> > >medium the better.
> > 	Ok.
> > >
> > >The second rule is that smaller specimens freeze better and the 
>out-most
> > >part of the specimen will be best preserved. Flat specimens are 
>suitable.
> > 	Small is lovely, but remember we are dealing with LM Histochemistry
> > and not EM. Orientation of a flat piece of TS muscle is a bummer, 'tall 
>and
> > slim or short and squat is in'
> > >
> > >Third, specimens with lower water contents will show less damage. So, 
>skin
> > >(or
> > >cork!) will be less affected by ice crystal formation than liver.
> > > Ice crystal damage can be reduced by fixing and then infusing with say 
>20%
> > >glycerin, but this may defeat your reasons for cryo.
> > 	Ok, but for muscle enzyme histochemistry a wee whiff of fixatives
> > is the kiss of death.
> >
> > >A very good freezing medium is liquid propane, cooled by liq nitrogen 
>like
> > >your
> > >isopentane.
> > >Use a fumehood throughout. Prepare your cold-cup with liquid nitrogen. 
>Have
> > >a
> > >needle (cut off square 19 gauge or similar) inserted into a bit of 
>tygon
> > >tubing
> > >connected to the outlet valve of a small propane gas cylinder (without
> > >regulators; purity of gas is not very important). Sit the cylinder
> > >up-side-down
> > >so liquid is expelled (preferred but works either way).
> > 	Propane is a good cryogen, but again this is muscle histochemistry
> > and not EM.
> > 	WITHOUT REGULATORS, in the name of the wee man, Safety Officers
> > would go totally ballistic without a back flow/flame regulator.  -180
> > atmospheric oxygen dissolves into the cold propane and what a 
>combination
> > that is and no regulator to stop the back flow into the propane tank. I
> > agree the purity of the propane is not important, in fact impurities are
> > good. I use a stirred mixture of isopentane and propane in order to 
>depress
> > the temperature and keep it away from -180 and the naughty effects of
> > oxygen.
> > >
> > >Open the valve very slowly so gas can only just be feeled on skin. Rub
> > >needle
> > >gently in the cold cup. The emitted gas will turn into liquid.
> > >Snap freeze specimens by very rapid immersion movement. The best 
>freezing
> > >happens in a fraction of a second and largely depends on the 
>temperature
> > >differential between specimen and the medium; since the interface warms
> > >rapidly, the rapid movement is required.
> > >Also assure that the transfer into liquid nitrogen for specimen storage 
>is
> > >very rapid.
> > 	Ok. Although I agree propane is very good for cryo EM, the best
> > cryo-preservation I have ever obtained was with a specimen slammed on a
> > cooled ultra pure copper block and freeze-substituted. I even published 
>a
> > micrograph in the Proceedings of the RMS so happy was I. Muscle
> > histochemistry, isopentane cooled with liquid nitrogen is the business.
> >
> > >Hope this helps.
> > >Cheers
> > >Jim Darley
> > >
> > >ProSciTech                 Microscopy PLUS
> > >PO Box 111, Thuringowa  QLD  4817  Australia
> > >Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
> > >Great microscopy catalogue, 500 Links, MSDS, User Notes
> > >                      www.proscitech.com.au
> > >> -----Original Message-----
> > >> From:	Tim Fairchild [SMTP:timf@cyllene.uwa.edu.au]
> > >> Sent:	Thursday, July 01, 1999 3:14 AM
> > >> To:	HistoNet Server
> > >> Subject:	Freezing muscle sections
> > >>
> > >> We have recently undertaken a project which required a portion of 
>muscle
> > >> to be analysed for fibre type and oxidative capacity.  The technique 
>we
> > >> adopted to freeze the muscle (human muscle), was to mount the muscle 
>on
> > >> cork using 'gum tragacanth', and then freezing this in isopentane 
>cooled
> > >> in liquid nitrogen.  The trouble we're having is that every 5th 
>sample
> > >> (roughly speaking) has ice crystal artifact through it.  I am
> > >> attributing this to the isopentan not being cold enough.  I guess my
> > >> questions therefore are:
> > >>
> > >> 1. Is there a way to protect the muscle from the freezing process, 
>i.e.
> > >> putting O.C.T. over the muscle?
> > >> 2. If the muscle has to be frozen in isopentane, what 'set up' has
> > >> worked for other people (i.e. we put the isopentane in a long metal
> > >> cylindrical container, inserted in a larger container holding liquid
> > >> nitrogen) and what techniques have you found useful (e.g. hold in
> > >> isopentane for 20 seconds)?
> > >>
> > >> Any help (or small tips) would be very much appreciated!
> > >>
> > >> Thanks in advance,
> > >>
> > >> Tim Fairchild.
> > >> -----------------------------------------------------------------
> > >> Timothy J. Fairchild B.Sc. (Hons)
> > >> PhD Candidate
> > >> Co-ordinator for Centre of Athletic Testing
> > >> Department of Human Movement and Exercise Science
> > >> Nedlands, Western Australia 6907
> > >> Telephone: (+61 8) 9380 2793
> > >> Facsimile:  (+61 8) 9380 1039
> > >> Email: timf@cyllene.uwa.edu.au
> > >> http://www.general.uwa.edu.au/~hmweb/index.htm
> > >>
> > >>
> > >>
> > >
> >
> > Dr. Ian Montgomery,
> > West Medical Building,
> > University of Glasgow,
> > Glasgow,
> > G12 8QQ,
> > Scotland.
> > Tel: 0141 339 8855 Extn. 6602.
> > Fax: 0141 330 4100.
> > e-mail: ian.montgomery@bio.gla.ac.uk
> >
> >
>
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 11:46:05 -0500
>From: "John Shaw" <jolesh@hotmail.com>
>Subject: Fwd: Position(s) Available
>
>
>
>
> >From: John Shaw <jolesh@hotmail.com>
> >To: HistoNet@Pathology.swmed.edu
> >Subject: Position(s) Available
> >Date: Sat, 19 Jun 1999 10:06:40 PDT
> >
> >Histonetters;
> >
> >I was asked to pass this on:
> >
> >Histotech position available, HT/HTL(ASCP)
> >M-F with rotating Saturdays Various shifts.
> >
> >Phoebe Putney Memorial Hospital is located in Albany, Georgia, a
> >fair-sized,
> >quiet city filled with Southern Charm.  Convienently located within equal
> >driving distance of Atlanta, the Gulf beaches, and the Atlantic coast.
> >For more details, please contact Lorraine Smallwood at 912-889-4124, or 
>Dr.
> >Laurence Danzer at 912-889-4028
> >
> >
> >Thanks guys,
> >J.Shaw, HT(ASCP)
> >
> >
> >_______________________________________________________________
> >Get Free Email and Do More On The Web. Visit http://www.msn.com
> >
>
>
>_______________________________________________________________
>Get Free Email and Do More On The Web. Visit http://www.msn.com
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 13:16:09 -0500
>From: "Ronan Ward" <rward@iol.ie>
>Subject: Re: Clearing agents and Immunocytochemistry
>
>Dear Ian,
>
>I've also seen very little on this topic.  One very detailed paper (the 
>sort
>your glad somebody else has done) was in the JCP in 1997 (Tissue 
>preparation
>for Immunocytochemistry --Williams, Mepham and Wright 50:422-428).  This
>covered all aspects from fixation through to section storage.
>
>In tissue processing they found that only temperature and the duration of
>the dehydration and wax infiltration steps had any affect on
>immunoreactivity.  They compared xylene, chloroform, clearene (Surgipath,
>uk) and Shandons xylene substitue.
>
>Overall the temperature and the duration of section drying had the greatest
>affect.
>
>Ronan
>
> >Date: 8 Jul 1999 10:07:01 -0500
> >From: Ian Montgomery <ian.montgomery@bio.gla.ac.uk>
> >Subject: Clearing agents.
> >
> > Clearing agents for tissue processing in immunohistochemistry.
> > Xylene is ok but what about the others, chloroform, benzene,
> >toluene, amyl acetate etc. I'm a wee bit suspicious about loss of
> >antigenicity with chloroform but nothing I'd like to stake the contents 
>of
> >my wifes wallet on.
> > Haven't found a lot in the literature regarding use, any thoughts.
> >Ian.
>
>
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 13:16:32 -0500
>From: "Ronan Ward" <rward@iol.ie>
>Subject: Re: Sakura coverfilm
>
>Hi,
>
>We have used the Sakura coverslipping film since early 1996.  Some of the
>coverfilm is now begining to lift from the edges (similar to the way
>mountant withdraws from the edges over time).  I have heard of other labs
>with slides only a little older having coverfilm lift completely taking the
>section with it.
>
>I'd be interested to hear the experiences of users of  the system for
>similar or longer periods.
>
>Ronan Ward
>Histopathology
>St. James's Hospital
>Ireland
>
>
>
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 16:31:27 -0500
>From: Linda Jenkins <jlinda@ces.clemson.edu>
>Subject: Removing yourself!
>
>Dear  "digest" users who would like to be removed from the HistoNet:
>	I sincerely hope that you get your name(s) removed from the
>HistoNet quickly as you doubled the space required to open my usual
>HistoNet mail this morning.  Why? Well...it could be that, as a "digest
>user" you REPLIED  to a HistoNet message without deleting the highlighted
>area, so ...your message contained all of the digest for that day. As a
>HistoNet digest user you shouldn't do this. ALWAYS send a new message to
>the HistoNet.  And remember - you are writing to a computer - not a person.
>Simply type "unsubscribe" (not unscribe, or unsribe, or one of the many
>variants we mortals manage to create) in the subject line.   It is not
>necessary to type anything in the body of the message.  Computers don't
>understand this and get especially confused by words like please and
>thank-you.
>	Hope this clears the confusion.  If not, there will be over a
>thousand? of us watching to see which one us misspells that
>subscribe/unsubscribe word again:-)
>			Linda
>
>
>*********************************
>Linda Jenkins, HT
>Clemson University
>Department of Bioengineering
>Clemson, SC
>**********************************
>
>
>----------------------------------------------------------------------
>
>Date: 11 Jul 1999 21:01:15 -0500
>From: Penelope Marr <MarrP@SESAHS.NSW.GOV.AU>
>Subject: RE: Clearing agents.
>
>Dear Ian,
>I'm not convinced one way or the other about the impact of different
>clearing reagents.  I have used several different clearing agents for
>various projects over the years but the only times I have had real
>trouble was when our satellite lab used "Histolene" (a limonene based
>product) and when "Mr Nobody" tampers with the tissue processors.  On
>one ocassion Mr Nobody forgot to change the processors' reagents for a
>fortnight which led to the same appalling IHC which resulted on tissue
>from the satellite lab.  The latter event has led me to wonder if the
>problem in the satellite lab was that the solutions were not changed
>frequently enough rather than histolene being the lousy option for
>clearing which I had first thought.  I suspect that people use the
>processing schedules which were designed for xylene and undenatured
>alcohol but have neglected to make the appropriate alterations in
>processing times and frequency of reagent changes to reflect the use of
>different clearing reagents and quality of alcohol.
>
>In short, I believe that the processing schedule plays a far more
>critical part in IHC than we acknowledge.  When Mr Nobody has changed
>our processing schedule or the grade or reagents I observe the results
>in the IHC long before any changes are noticed in the morphology, H&E
>staining or routine special stains.  I wish I had more time to explore
>the topic further.
>
>Penny Marr
>
>
> > -----Original Message-----
> > From:	Ian Montgomery [SMTP:ian.montgomery@bio.gla.ac.uk]
> > Sent:	Thursday, 8 July 1999 3:26
> > To:	HistoNet@Pathology.swmed.edu
> > Subject:	Clearing agents.
> >
> > 	Clearing agents for tissue processing in immunohistochemistry.
> > 	Xylene is ok but what about the others, chloroform, benzene,
> > toluene, amyl acetate etc. I'm a wee bit suspicious about loss of
> > antigenicity with chloroform but nothing I'd like to stake the
> > contents of
> > my wifes wallet on.
> > 	Haven't found a lot in the literature regarding use, any
> > thoughts.
> > Ian.
> >
> >
> >
> > Dr. Ian Montgomery,
> > West Medical Building,
> > University of Glasgow,
> > Glasgow,
> > G12 8QQ,
> > Scotland.
> > Tel: 0141 339 8855 Extn. 6602.
> > Fax: 0141 330 4100.
> > e-mail: ian.montgomery@bio.gla.ac.uk
> >
> >
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