I am currently trying to section FFPE mouse brains using a rotary microtome for fluorescence IHC.
I'd prefer to collect the sections free floating in buffer, stain them free floating and then mount them on slides after staining. However, when I tried to deparaffinize 25 micron thick free floating sections, the xylene washes made my sections way too fragile and they began to tear really easily.
Would it be worth trying to section at 40 microns, in hope that xylenes won't damage thicker sections as much, or should I give up on collecting the sections free floating and just collect my sections mounted on slides? If mounting is the best solution, what is the thickest sections that are possible for F-IHC of slide mounted sections?
Thanks in advance for any advice!
The biggest thing in this question that stuck out to me was de-paraffinizing free floating sections. You cannot do it. In removing the paraffin from the section with xylene, you are not replacing it with water. It is a brittle section and will shatter upon manipulation. I have had sections slide off a slide during dehydration in xylenes and they are absolutely stiff.
Collect your paraffin sections on slides and then stain. 20 um should be thick enough for staining on slides although they might end up being a little thicker in the end after rehydration. The general rule is that antibodies have 20 um of penetration per side. 40 um is too thick for efficient penetration on slide based staining (hence free floating) without some sort of weird quirk like adding DMSO to your primary to increase penetration or just using it at an increased concentration to compensate.
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