Hi,
Does anyone have experience doing immunofluorescence on unfixed embryonic (E18.5) cryosections? I usually postfix my sagittal cryosections using 4%PFA for 10 minutes then continue a "standard" IF protocol, but I have noticed that the brain and spinal cord still seem to come off. Any tips?
Does the neural tissue come off while doing the IF , or during the cutting?
18.5 embryos are almost newborns, so bones might be difficult to cut off, so neural tissue is maybe ripped off and you might consider to buy stronger blades.
If you are losing it during the IF protocol, and through the several washes, you could try Gelatin Coated slides as the charges interaction could not be strong enough.
The only problem is that sometimes, you got autofluorescence due to Gelatin, just give a try.
Good luck!
I think that the period of postfixation is still shorter. May be experiment 2-3 other longer periods
1)Look into: A.C.Cuello Immunohistochemistry IBRO Handbook series: Methods in the neurosciences volume 3.chap 14 and 15
2) coat your glass
I cut unfixed mouse embryonic organ culture sections (genital ridge) at 8um on electrostatic charges slides. The only clear difference was that unlike fixed sections, the unfixed don't cope well with storage at -20 so I just cut what I needed at the time and stored the uncut unfixed remains in an air tight container.
Perhaps the sections are not completely air dry before processing? The use of a fan can help prevent any accidents !
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