[Histonet] Formalin-fixed paraffin embedded question

[sgoebel <@t> mirnarx.com]

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
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>