[Histonet] P21 mouse brain

[Geoff McAuliffe]

I think your processing schedule is too short for a whole mouse brain. 
Do you really need to cut the entire brain in one block?
I suggest cutting the brain in half down the midline or in thirds 
rostral-caudal and doubling or tripling the times in all steps. Also add 
a 100% alc:xylene 1:1 mix step.
A third paraffin is also a good idea, especially since you don't have 
vacuum.

As to the problems with cryo sections, first wash the brain in buffer, 
then try 10% sucrose cold, then 25-30% cold over night.
How are you freezing the brains? Just putting them in the cryostat is 
not sufficient, they must be completely frozen very quickly, then 
allowed to equilibrate in the cryostat.
-20C should be fine, is your knife sharp?

Geoff

On 1/23/2013 12:54 PM, Papke, Louisa M. wrote:
> 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
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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
**********************************************