[Histonet] PFA: how long per mass/type tissue?

[Tyrone Genade]

Hello,

Is there a guideline as to how long to fix in PFA based on the mass of
the tissue or type of tissue?

I am working with fish. I have had been experimenting with Davidson's
Fixative but it gave too much background under the fluorescence
microscope. I have tried fixing in PFA and then decalcifying in
Kristenson's and this worked very well but I have lost some antigens.
I know, from past experience, that a fixing the dissected out brain in
PFA for 10 minutes on ice did not destroy the antigens so I am now
wonder if the problem is over-fixation? What I did this time round
(because it is very difficult to dissect out the entire brain without
losing the hypothalamus and damaging the tectal surface) was to cut
away the excess tissue and fix the cranium with eyes overnight at 4 oC
in PFA and the decalcify in Kristenson's for 2 days at 4 oC. I got
good cryo-sections and the fine structure was well preserved.

The cranium is small, about 12 x 6 mm in total and about 4 mm thick,
so I guess a mass of about 200 mg. I'm going to try a 2 hour fix* at 4
oC and see how that goes. Any suggestions? I don't want to kill fish
needlessly.

*I have read of perfusion fixed rat brain being post-fixed for two
hours at 4 oC and the published sections look quite good. I can't
perfusion fix. I can't find a vessel large enough for a canula.

Thanks

-- 
Tyrone Genade
http://tgenade.freeshell.org
email: tgenade <@t> gmail.com
tel: +27-84-632-1925 (c)
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