Re: Rat Tissue Processing-II

From:Nancy E Weber <>

Hello Cyrla,

When I worked at a vet. diagnostic lab in Washington, we had problems with
the pathology residents cutting brain tissues too thick. Since a good bit of
brain is water the thicker sections did not dehydrate properly in the
regular processing cycle that all tissue went through. We had to get them to
cut them thinner or they had to process longer using another processor that
we used to have set up for whole sections of brains of larger animals. These
under processed brains would just disinergrate upon hitting the waterbath.
3mm sections might be too thick for the processing schedule you have set up.
Also, as others have pointed out, turning the waterbath down helps

----- Original Message -----
From: "C H" <>
To: <>
Sent: Monday, April 23, 2001 2:59 PM
Subject: Rat Tissue Processing-II

> 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
> and spinal cord tissue.  I understand that CNS tissue, both human and rat,
> require different schedules than "peripheral" tissues in order to have
> 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
> 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
> 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
> 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
> 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,
> 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.
> Cyrla
> >From: C H <>
> >To:
> >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
> >right combination of steps to prevent the tissue from overly expanding
> >separating from the paraffin in the water bath.
> >
> >Any assistance would be greatly appreciated.
> >
> >Cyrla
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