[Histonet] Formalin-fixed paraffin embedded question

[Rene J Buesa]

According with your protocol it seems that you are processing by hand. Even so, the periods seem too long, specially for brain using xylene.
The best (and "final") solution for your problem will be to abandon xylene and start using mineral oil.
René J.

--- On Mon, 2/21/11, Noel Gray <GrayN <@t> upstate.edu> wrote:


From: Noel Gray <GrayN <@t> upstate.edu>
Subject: [Histonet] Formalin-fixed paraffin embedded question
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, February 21, 2011, 3:54 PM


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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