[Histonet] Formalin-fixed paraffin embedded question
Grantham, Andrea L - (algranth)
algranth <@t> email.arizona.edu
Mon Feb 21 16:25:22 CST 2011
I agree. Your tissue isn't fixed well enough and the dehydration steps seem long, especially since at some point you split the brain and the spinal cord. After perfusion immerse in 10% NBF (a few changes) at least 24 hrs (I like to leave it in formalin longer). If you are fixing the brain whole remember that formalin is a slow fixative and it takes some time to penetrate all the way into the brain even if it was perfused first. I found that by combining perfusion with immersion in the fixative the brain fixed and cut better. Before you start dehydrating, try washing the tissue in running water at least an hour (or longer). Then start your dehydration.
Xylene makes tissue brittle and overnite in xylene I think isn't doing you any favors. I use Clear Rite 3 for my clearing agent.
Are you hand processing? Do do have access to vacuum infiltration? I have a protocol for mouse brains but it is for processing on a VIP.
When cutting try soaking the block at room temp in water to which a bit of glycerin has been added. I usually don't place the brains on ice but just in cool water and my room is always cold.
I read here on histonet that people let their tissues soak for a few minutes but with mouse tissue you need to soak it longer. I don't really time the soaking but it is way longer than a few minutes. Then you might try wiping the surface of the block with a gauze sponge or even a cotton tipped applicator that was dipped in the soaking water in between sections as you are cutting your ribbon - don't make it too wet just moist.
On Feb 21, 2011, at 1:54 PM, Noel Gray wrote:
> 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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