[Histonet] Formalin-fixed paraffin embedded question

[Grantham, Andrea L - (algranth)]

The NSH Manual needs to be seriously updated. There is no protocol for mouse brain that could help this guy with his problem.

Andi



On Feb 22, 2011, at 2:02 AM, Margaret Blount wrote:

> 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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