[Histonet] Re: (no subject) (Geoff McAuliffe)

[Jean-Martin Lapointe]

Tojo,
Geoff provides very good pointers, but I think his option 3. is the correct one in your case, i.e. what looks like 'holes' are areas of glycogen and/or lipid that are unstained with the technique you are using. If your rats are not fasted prior to euthanasia, the cytoplasm of the hepatocytes will be filled with glycogen. Trying a PAS would likely confirm this, by staining the clear areas positive.

Jean-Martin

__________________________________
Jean-Martin Lapointe, DMV, dACVP
AccelLAB Inc
 


------------------------------
Date: Tue, 16 Sep 2008 14:17:48 -0400
From: Geoff McAuliffe <mcauliff <@t> umdnj.edu>
Subject: Re: [Histonet] (no subject)
To: "TOJO (Torben Seested Johansen)" <tojo <@t> novonordisk.com>

Dear Tojo:

    Your problem could be due to 3 things. 1. Waiting too long to fix 
the tissue. 2. Freezing too slowly. 3. Interpreting the lack of staining 
of hepatocyte glycogen as an artifact.

1. You are waiting 5 minutes after death to fix the tissue. There is 
absolutely NO reason to spend 5 min pumping 100 ml of PBS through the 
circ. system. You will never wash out all of the blood and there is no 
need to. AND when you finally get to fixative, the flow of fixative is 
too low. Here is the solution to your problem.

Use a 16 g needle in the L. vent.
Pump 10-15 ml of room temperature PBS into the rat in 15-20 seconds.
Pump 300 ml of room temperatue fix in 5 minutes. The fix is 4% 
paraformaldehyde for light microscopy. For transmission EM the fix is 2 
or 3 or  4% paraformaldehyde with 1% glutaraldehyde. Buffer can be 
either phosphate or cacodylate. The advantage of cac. buffer is that you 
can add some calcium salts to stabilize membranes for EM (2milleMole) 
without percipitation.Yes, you can add Ca salts to phos. buffer but it 
will precipitate instantly (look up the solubility of CaPhosphate, or 
should I say the insolubility). Of course, cacodylate has arsenic in it 
so don't lick your fingers.
Remove liver and cut into appropriate sized pieces. For light microscopy 
fix as long as possible, formalin reacts with tissue very slowly. 
Glutaraldehyde fixed much faster, an hour or two is plenty.
Cryoprotect with sucrose.
2. Tissue must be frozen VERY QUICKLY with 2-methylbutane (isopentane) 
cooled with dry ice or liquid nitrogen otherwise you will have large 
holes due to ice crystal formation.
3. The stain you are using, Tol.Blue will not stain glycogen so you may 
have empty-looking areas because of this. Use Periodic acid Schiff for 
glycogen but NOT after glutaraldehyde fixation.

Geoff


TOJO (Torben Seested Johansen) wrote:
> 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