RE: [Histonet] Soaking paraffin blocks

From:"Nicola Cragg"



Hi Kemlo,

Thanks for the message and explanations - sorry for the slow response -
life is very hectic in contract research particularly in a relatively
new biotech company - I don't recommend it.  The company arose out of
the Department of Epithelial Biology at the Paterson Institute for
Cancer Research at Christies Hospital and much of the company's work /
assays are based on the Prof. Potten's work on the gut (the website will
tell you much more).

I'm ok with the poor fixation.  The majority of our gut samples are
fixed in Carnoy's (contains 60% absolute alcohol)for 1 hour and then
transferred to 70% alcohol prior to processing. This method has been
used for about 30 years, way before my time. 

What do you mean when you say "If the processing fluids are left on too
long, tissue goes hard" - are you referring to too much dehydration or
that the fluids have been left on the processor too long?  After a
previous discussion on Histonet, I had understood that some think it's
not possible to over dehydrate - do you not agree?  The schedule I use
is quite lengthy so I don't think it can be a case of too little
dehydration. 

With the xylene and wax stages, I thought I had encorporated
sufficiently long incubation times to ensure clearing and wax
penetration of tissues.  I have wondered whether my timings are too long
and have cut back on the wax times, which has made no difference to the
sectioning.  Plus I regularly change my reagents and probably do this
more than necessary, so I don't think it's too much carry over.  I will
think more about the schedule though.

I do think the nature of the samples has a lot to do with it and as I
mentioned before an average block can contain up 96 pieces of intestine
orientated on end and it seems to me that cutting through all those
different layers of muscle, mucosa and epithelium repeatedly through the
block could be the root of the difficulties - although I can cut them
fine after a few minutes on wetted ice.

It's been an eye-opening thread and it's this kind of thread which
challenges the routine methods you do every day without thinking which I
think help one become a better histologist, well hopefully anyway!!

Thanks,

Nicola

-----Original Message-----
From: Kemlo Rogerson [mailto:Kemlo.Rogerson@waht.swest.nhs.uk] 
Sent: 29 October 2007 13:40
To: Nicola Cragg; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Soaking paraffin blocks


"What am I concerned with is the claim of poor processing - how does one
know that it is poor processing, particularly if you haven't seen the
blocks or attempted to cut them?  What is poor about it - insufficient
wax penetration, lack of dehydration?  Processing is essentially quite a
straight forward process and apart from varying timings in alcohols,
xylene and wax, I can't understand how it could go so wrong?!!  I have a
Leica TP1020 without vacuum and run overnight schedule based on what
I've read up - reagents are changed regularly always erring on the side
of caution.  

I am a self-taught histologist (and there may lay your accusations -
LOL) and have been "practicing" for over 5 years now, but I have only
learnt by reading up on what the experts say and do (including Histonet
- which I have found to be a fantastic tutor) and by practice.  I am
always keen to learn from my more experienced peers so please expand on
pharses such as "poor processing" and "poor fixation"."
Nicola

Poor fixation could mean not enough or too much fixation. Some fixatives
harden tissue when 'overexposed' some fixative take some time to
penetrate and to fix. Bluntly, IMHO, you can't process properly unless
you've fixed 'properly'. Fixation puts the tissue in the correct state
to resist the effects of processing and to allow the fluids to
penetrate. 

If the processing fluids are left on too long, tissue goes hard, too
little and water or alcohol isn't removed. If the tissue isn't clear in
xylene then it's not dehydrated, if it's not dehydrated then you won't
get the wax in properly, if you don't get the xylene out then you make
the wax and the tissue 'soggy'. Overprocess and you harden the tissue
(usually the heat from the wax) and you have to use reclamation
techniques such as ice/ water IMHO. You build a house from the
foundations up; fixation is your foundation without which nothing
underpins the processing.

What does your Company do in the UK?

Kemlo Rogerson
Pathology Manager
DD   01934 647057 or extension 3311
Mob 07749 754194; Pager 07659 597107;
Never does nature say one thing and wisdom another. --Juvenal 

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