Hi again,

 

I apologize for not giving the recipe for the fixative I was using. It is the zinc salt fixative from the 1994 Beckstead paper (J. Histochem Cytochem 42: 1127-1134). The composition is as follows:

0.1M TrisHCl (pH 7.4) with 0.05% Ca acetate, 0.5% Zn acetate and 0.5% Zn chloride.

The final pH of this fixative ends up being around 6.8, but you are warned not to try to bring it closer to neutral pH as precipitates will form.

Initially I tried clearing the blood from the mouse with 0.1M phosphate buffer. However, as soon as I started running the zinc fixative through the tubing (i.e. before it even got into the animal), a white precipitate formed in the tubing - and i imagine also in the animal. I assume this was due to the zinc fixative coming into contact with phosphate molecules left in the tubing from the previously run phosphate buffer.

I next tried clearing the animal with 0.1M Tris base buffer, pH7.4. I did not see any precipitation form in the tubing, and have no idea what was happening in the actual animal. I wasn't sure though if the Tris was a good buffer with which to clear out the blood in the animal.

So, my questions are:

1). Is this zinc salt fixative a good fixative for doing whole animal perfusions?

2). If it is, what buffer would you suggest for clearing the animal with prior to fixation

3). How long would you post-fix an adult mouse brain for, and would you recommend trimming the brain to the smallest possible size (i.e. only taking the part in which I am interested) so as to decrease the time needed for post-fixation?

My end goal here is to use the Pharmingen mouse anti-mouse I-Ab antibody to localize MHC II molecules in the mouse brain. Pharmingen recommends the use of a non-formalin, zinc fixative as the MHC molecules are known to be difficult to localize with immunohistochemistry in paraffin embedded tissue

 

Hope this makes sense. Thanks for any and all advice you can offer.

 

Andrea Portbury

Neuroscience Center

University of Chapel Hill, NC.