RE: [Histonet] mouse heart chatter upon sectioning...

From:"Tony Henwood"



Ice crystals can form at"4oC" - Fridge temperatures will vary, often colder than the stated temp, often warmer.
I did not suggest that ice crystal formation would cause shatter but it will effect morphology (see 
Henwood, A., (2005) "Effect of Slide Drying at 80C on Immunohistochemistry" J Histotechnol 28(1):45-46.).

Some researches (and I say SOME not all), often with limited understanding of the processes of histotechnology, will sprout expertise and knowledge that is based on flimsy evidence. Fixing in formalin seems to be one of these. Someone decided in their wisdom to fix at 4oC. Why?, Based on what? So others thought it sounded good to do it this way and problems have occurred ever since.

Preventing over-fixation in formalin will invariably cause over-fixation in ethanol.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) 
Laboratory Manager & Senior Scientist 
The Children's Hospital at Westmead, 
Locked Bag 4001, Westmead, 2145, AUSTRALIA. 
Tel: 612 9845 3306 
Fax: 612 9845 3318 


-----Original Message-----
From: Gaupp, Dina D [mailto:dgaupp@tulane.edu] 
Sent: Thursday, 5 June 2008 9:38 AM
To: Tony Henwood
Subject: RE: [Histonet] mouse heart chatter upon sectioning...


Fix @4C to prevent overfixation.  I didn't think ice crystals could form @4C.

Process temps are all performed at room temp. except of course, paraffin tremp is 57C.

Process times vary according to size & complexity of tissue.  The last process time for mouse hearts was total of 5hrs.  But it still didn't work.  The tissue still chattered.  I had to soak the block in ice cold water to soften the tissue.  What schedule would you recommend processing mouse hearts?  Alot of the principle investigators like to leave their tissues at 4C in fix.  Does this form ice crystals?  Only thing is every other tissue besides hearts cut perfect.  So I wouldn't think it would be ice crystals making it chatter.

Dina





From: Tony Henwood [mailto:AnthonyH@chw.edu.au]
Sent: Wed 6/4/2008 6:23 PM
To: Gaupp, Dina D ; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] mouse heart chatter upon sectioning...


Several queries,

Why fix in the fridge? Do you want to slow down the fixation or
introduce ice crystal artifact?

If the tissue is shattering, one of the problems might be the fact that
the tissue is fixed in ethanol reather than formalin.

What are the processing times & temperatures.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Gaupp,
Dina D
Sent: Thursday, 5 June 2008 9:16 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] mouse heart chatter upon sectioning...


Whenever I process (whole)mouse hearts, the tissue chatters upon
sectioning.  The tissue is usually fixed overnight in 10%nbf at 4C
before processed.  I tried different processing times.  The last time I
processed hearts, I processed them for 5 hours total no vaccum.  Should
I decrease the processing time even lower than that?  I am afraid of
underprocessing.  I have to section the entire heart from top to bottom,
so I really need some advice.

(open system) Processor Set Up:
1 - fix (if tissue fixed overnight, skip #1 & #2 stations)
2 - fix
3 - 70%
4 - 80%
5 & 6 - 95%
7 & 8 - 100%
9 & 10- Xylene Sub. - Clear-rite
11 & 12-Paraffin stations

Thanks,
Dina
dgaupp@tulane.edu 
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