[Histonet] Formalin-fixed paraffin embedded question

[Margaret Blount]

I agree with all the comments so far, and I think all the steps are far
too long, particularly the overnight in molten wax. You are effectively
cooking your samples. I would avoid overnight in 100% ethanol too; it
will tend to harden the tissue.
Get hold of the manual for animal tissues available from the Society for
histotechnology. I found this invaluable and now most of my tissues can
be sectioned without soaking at all. I just chill them slightly, if
necessary.

Good luck with your samples.

Margaret

Miss Margaret Blount
Histology Manager
Metabolic Research Laboratories
Level 4 Institute of Metabolic Science
Box 289, Addenbrooke's Hospital
Hills Road, Cambridge, CB2 0QQ

Tel 01223 769061/336079


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Noel
Gray
Sent: 21 February 2011 20:55
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Formalin-fixed paraffin embedded question

I am having an issue with formalin-fixed, paraffin embedded tissue that
I am
sectioning. I am using a microtome to cut the tissue (mouse brain,
cerebellum and spinal cord) in 10 um sections which I will stain using
cressyl violet. Sometimes, the tissue in a block will splinter once it
hits
the blade. Usually all samples from the same animal splinter but this is
not
always the case. If I put the block into the water bath (sectioning
surface
exposed to water or not) this seems to stop the splintering for 1-200 um
worth of tissue. However, I am afraid this may bring error into the
histological analysis of my tissue.
 
I assume it has something to do with the protocol I am using to prepare
the
tissue. I guessed that maybe the dehydration, alcohol clearing, or
paraffin
infiltration are not complete, resulting in the problem I have. However,
I
looked at various FFPE protocols and each of my wash steps are longer,
which
may be the problem? I was wondering if anyone has encountered this
before,
or if anyone knows exactly what is going on with my tissue and how I can
fix
it? Thank you. 
 
Here is my protocol:
 
-Anesthetize mouse followed by a system flush of 30 ml of PBS and then
slow
perfusion of 50 ml of 4% PFA.
-Brain and spinal cord are removed as a single, in tact unit and placed
into 70% ethanol for 4 hours
-80% EtOH for 4 hours
-90% EtOH over night
-100% EtOH #1 for 4 hours
-100% EtOH #2 for 4 hours
-100% EtOH #3 over night, forebrain cerebellum and spinal cord are
separated
-Xylene wash #1 for 4 hours
-Xylene wash #2 for 4 hours
-Xylene wash #3 over night
-Moulton paraffin wash #1 for 4 hours
-Moulton paraffin wash #2 for 4 hours
-Moulton paraffin wash #3 over night
-Tissue is embedded and then sectioned into 10 um sections
 
Again thank you for your time.
 
Noel W Gray
Neuroscience Graduate Program
SUNY Upstate Medical University
3219 Weiskotten Hall
766 Irving Ave
Syracuse, NY 13210-1630
(315) 464-8144
grayn <@t> upstate.edu
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