[Histonet] P21 mouse brain

[Papke, Louisa M.]

The mice were perfused with 4% PFA.  Then left in 4% PFA for about 2 weeks.  
The processing schedule is as follows:
70% Alcohol   15 min.
80% Alcohol   15 min.
95% Alcohol   15 min.
95% Alcohol   15 min.
100% Alcohol 15 min.
100% Alcohol 15 min.
100% Alcohol 20 min.
Xylene  20 min
Xylene  20 min
Xylene  45 min
Paraffin 30 min
Paraffin 40 min.    temp 58 degrees.  No vac. on any of the stations.

This is the same program I use on mice spinal cords. 
Half the mice are under hypoxic conditions and the other half normoxic conditions.  The normoxic brains also fell apart.   
I also did another group with the perfusion with 4% PFA then cryoprotected with 30% Sucrose.  They didn't cut well.  Wrinkles and air bubbles.  I will try cutting at a -25 instead of -20. Also, the sections fell off as soon as I put them in water. 

I will admit that I have only worked with adult mouse brain and spinal cord.  So this is a new area for me.

Thank you again for suggestions and advice.
Louisa M. Papke
Research Technologist
Demyelinating Diseases Research

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff McAuliffe
Sent: Wednesday, January 23, 2013 11:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] P21 mouse brain

On 1/23/2013 11:10 AM, Papke, Louisa M. wrote:
> Has anyone worked with P21 mouse brain?  Such as frozen or paraffin sectioning?  Right now after perfusion and paraffin embedding, the brain literally falls apart when picking it up to embed.  Any ideas or hints available would be welcome.
>
> Thank you in advanced,
> Louisa M Papke
>
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Perfusion with what? Fixation for how long? What is your processing schedule?

Geoff

--
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583
mcauliff <@t> umdnj.edu
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