[Histonet] Formalin-fixed paraffin embedded question

[Grantham, Andrea L - (algranth)]

I'd be happy to help. Maybe you can too! 8-)



On Feb 22, 2011, at 8:53 AM, <sgoebel <@t> mirnarx.com> wrote:

> Are you volunteering?  That is so nice of you =)
> 
> Sarah Goebel, BA, HT(ASCP)
> Histotechnologist
> Mirna Therapeutics
> 2150 Woodward Street
> Suite 100
> Austin, Texas  78744
> (512)901-0900 ext. 6912
> 
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> Grantham, Andrea L - (algranth)
> Sent: Tuesday, February 22, 2011 9:48 AM
> To: Margaret Blount
> Cc: Noel Gray; histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] Formalin-fixed paraffin embedded question
> 
> 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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> 
>