[Histonet] Formalin-fixed paraffin embedded question

Noel Gray GrayN <@t> upstate.edu
Mon Feb 21 14:54:46 CST 2011

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
-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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