question regarding IF viewing in frozen liver - (Sep/26/2006 )

I used an aav to express GFP and RFP in order to look at co-localization in hepatocytes. When I removed the livers I could see the GFP by eye (the livers looked moldy). I cryosectioned the livers and then coverslipped them in a fluorescent mounting media but when I look on the scope I'm not seeing anything....

Do I need to fix them before coverslipping? If so, would cold methanol work or should I use a fomaldehyde fix?

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we fix tissues (4% paraformaldehyde) before sectioning them.

I have seen GFP flurescence in liver (infected by AAV) after fixing it and sectioning.

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these were fresh frozens.....imbedded in OCT medium and flash frozen

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I havent tried it this way.

after sectioning without fixing, if one washes the section, may be GFP might leak out. Just a guess.

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theoretically there's no washing required.....cut the tissue, mount on a slide and cover slip with Fluoromount G.

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this is new to me. I was told to always wash after cryosection.

is there a possibility that the sections u have mounted dont have the virus. try sections from a dif. area of the liver.

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