[Histonet] Mouse Brain Processing Help

[saby_joseph_a]

I agree with Liz's processing suggestions.  I would also suggest holding the brains in NBF, as holding rodent brain in 70% alcohol can cause microvacuolation in certain areas of the brain .
Joe Saby, retired. 


Happy Connecting. Sent from my Sprint Samsung Galaxy S® 5

-------- Original message --------
From: Elizabeth Chlipala via Histonet <histonet at lists.utsouthwestern.edu> 
Date: 12/02/2015  1:10 PM  (GMT-05:00) 
To: Laura Tarwater <tarwater.1 at nd.edu>, "'histonet at lists.utsouthwestern.edu'	(histonet at lists.utsouthwestern.edu)" <histonet at lists.utsouthwestern.edu> 
Subject: Re: [Histonet] Mouse Brain Processing Help 

Laura

They may not be processed enough.  We process brain samples for both mouse and rat 2 mm thick sections (cut on a brain matrix) on a 45 minute cycle.  If they are processing half of the mouse brain (these samples can be 4 -5 microns thick).  I don't think the cycle is long enough, that is why the blocks are swelling up when they are trimmed and placed on ice.  A well processed brain block should not swell up when it is placed on ice.  The separation of the brain tissue from the paraffin on the water bath is also a sign that the samples are under processed.

Here is the cycle we use for brain samples - you may need to adjust this a bit for your samples but I think if you process a bit longer they might have better success with sectioning.  Brain can be tricky.  We have the ability to add an extra 100%, xylene, and paraffin step to this cycle if necessary.  For mouse tissue (unless its skin, bone and brain) our cycle is normally 20 minutes per station,  but we have found that brain does need to be processed a bit longer.  

50% alcohol - 45 minutes
70% alcohol - 45 minutes
80% alcohol - 45 minutes
95% alcohol - 45 minutes
100% alcohol - 45 minutes
100% alcohol - 45 minutes
Xylene - 45 minutes
Xylene - 45 minutes
Paraffin - 45 minutes
Paraffin - 45 minutes
Paraffin - 45 minutes

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: Laura Tarwater via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Wednesday, December 02, 2015 10:40 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Mouse Brain Processing Help

I need some advice on processing mouse brain.  We have an outside lab using our automatic processor and embedding equipment and has been having issues section their mouse brain.  I was informed that the sections are extremely brittle and rolling but mostly just the paraffin and not the tissue itself.  They are seeing the brain tissue separate from the paraffin once it is the water bath.

Had better results when placing a soaked gauze on the surface of the block instead of chilling or soaking block to prevent swelling of tissue.

Here is the protocol they are using for harvesting and processing:
1.  Tissue perfused to 4% PFA at time of sacrifice
     Brain is cut in half at this point and separated 2.  Tissue placed in cassette in 10% NBF for 24hrs 3.  Then placed in 70% ETOH until able to process (usually the next day) 4.  Processing Schedule (all done with vacuum)
   30min 70%  ETOH
   30min 80%  ETOH
   30min 90%  ETOH
   30min 100% ETOH
   30min 100% ETOH
   30min 100% ETOH
   30min xylene
   30min xylen
   5min empty tank to remove excess xylene
   30min paraffin
   30min paraffin

I am not a histotech so any advice you can give me would be great.

Thanks,


*Laura Tarwater*

*Tissue Bank Technician*

*Harper Cancer Research Institute*


*University of Notre Dame574-631-2562*
*tarwater.1 at nd.edu <tarwater.1 at nd.edu>*
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