[Histonet] (no subject)

[TOJO (Torben Seested Johansen)]

Hi,
I do not seem to be able to achieve acceptble morphology of perfused rat liver. I seems as if some of the cytosol of the hepatocytes are "washed away" as seen in toluidine blue stained cryo-sections (see image18.jpg). I am to use the tissue cryo-sections in immunohistochemistry and transmission electron microscopy.

I my attempts to get an acceptable/good morphology I have tried fixation buffers consisting of 2, 4, 6 or 8 % paraformaldehyde (w/wo 0.1% glutaraldehyde).

My fixing procedure is as follows (all at room temperature);

a rat (~250g) is anasthestized and a perfusion needle is inserted into the left ventricle. The atrium is cut and PBS is flushed thru the rat at 20ml/min for 5min. The rat is thereafter fixated at 10ml/min for 10 min with fixation buffer (I have so far tried 2, 4, 6 or 8 % paraformaldehyde (with and with out 0.1% glutaraldehyde) in 0.1M cacodylatebuffer (pH 7.4) all with same unacceptable result (see image18.jpg). The liver is cut into smaller pieces (~2*2*2mm) post fixed for 1h in the respective fixative, transferred into 2.3M sucrose for minimum 1h  (preferably over night) and frozen in liquid nitrogen.

Any idea why teh cytosol of my hepatocytes seems to be "gone" (hydropic degeneration?) ?

I have searched the net and it seems as if theres a 1000's of different ways of performing rat liver perfusion fixation. Some use cold buffers and others also perfuse with sucrose solution (either in combination with the fixative or with sucrose alone post-fixation)

what can I do to try and optimise my perfusion ?

______________________________________

Torben Seested Johansen
Post Doc
Exploratory ADME, Biopharmaceuticals

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