Rat Tissue Processing-II

From:C H <cryla@hotmail.com>

Hello, Histonetters

Thank you to everyone who responded to my posting of last Friday. My 
apologies for being so vague. In my attempt to be brief and not bother you 
all with details, I guess I managed to be, well, too brief!

Allow me to clarify a few things. My main concern is processing of rat brain 
and spinal cord tissue.  I understand that CNS tissue, both human and rat, 
require different schedules than "peripheral" tissues in order to have ideal 
tissue preservation. I have no real details of a processing protocol of my 
own to share since I have only tried it twice, 2 different ways. ALSO, I 
failed to mention the very important point that I need to use this tissue 
for immunolabeling and in situ hybridization, not just histology.

Regarding the size of the tissue, I processed rat brain that has been 
perfused and left in fixative either all day or overnight at 4 degrees C and 
then cut into 3mm thick blocks. I have tried 10% NBF as well as 4% 
paraformaldehyde, I would prefer the latter. Once the cassettes were placed 
in the processor, the tissue sat in 10% NBF for 2 hours before continuing 
with the rest of the steps.

Let me also clarify that I think my waterbath temperature is fine (40degC). 
We have some rat brain that was processed and embedded for us by a CRO. We 
used these blocks as a starting point for our new "paraffin endeavor" and 
when I cut those, they float just fine. Upon asking them for their 
processing protocol all they would tell is is "it takes a long time, 
approximately 24 hours". Basically, they will not divulge their trade 
secret. It is unfortunate since the brains look very good, cut well, float 
well etc.  So, I am using these brains as a "control" if you will for my own 
processing.  Also, the tissue preservation in these blocks is much better 
than in sections of my own blocks.  Mine have some holes and the edges, the 
cortex, appears dried up and rough.  Perhaps it is the fixation time as 
Jeniffer Philopena suggested, or the temperature of the paraffin as Connie 
McManus pointed out.

I hope this clears a few things up. Thanks again.


>From: C H <cryla@hotmail.com>
>To: histonet@pathology.swmed.edu
>Subject: Rat Tissue Processing
>Date: Fri, 20 Apr 2001 17:22:24 -0400
>Hello, All
>I wonder if any of you have a protocol that you wouldn't mind sharing for
>auto-processing rat CNS tissue for paraffin embedding.  I am a novice at
>working with paraffin,using a Leica TP1050 and so far I have not found the
>right combination of steps to prevent the tissue from overly expanding and
>separating from the paraffin in the water bath.
>Any assistance would be greatly appreciated.
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