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Fixing tissue for cryosectioning

Immunofluorescence Fixing

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#1 HirubhaiAmbani

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Posted 16 August 2011 - 05:40 PM

Hello,

I have a question regarding fixing tissues for cryosection. I know that one can directly freeze and embed tissues in O.C.T, cut and stain it (immunofluorescence). However, i need good morphology and hence this method won't give me best results.

So I am going the other route, fix tissues in PFA, cryoprotect in 30% sucrose and then embed in OCT. Currently I am facing problems with sucrose. I know that the tissue sinks in sucrose solution after a day which is an indicator that the tissue is ready. I used to do this technique for liver and pancreas earlier and the tissue used to sink in the sucrose. However, this time I am working with breast tissue and it refuses to sink in 30% sucrose, even after 3 days. Should I just go ahead and embed the tissue in OCT and section it? Does somebody have some other suggestions? I dont have more mice (hard to breed) so I don't want to screw up the samples I already have.

Thanks a lot.

Regards,
Baba.

#2 bob1

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Posted 16 August 2011 - 05:52 PM

Breast tissue is largely fat and as such is very low density - it should float even when saturated with sucrose The fat is enclosed inside cells, but I don't know if this will affect how the sucrose permeates the tissue.

Why don't you just mount in parrafin and section from there, if you want good morphology?

#3 blablabla

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Posted 07 September 2011 - 08:35 AM

I think my problem relates to this area so I'm posting it in here rather than starting a new topic.

I need to cryosection trabecular bone for imaging of tetracycline labelling. However I also wish to perform a micro CT scan of the same bone sample. The nearest micro CT scanner is a 6-7 hour round trip (including approximate scanning time) so what I'm proposing is to fix the bone sample in formalin, perform the scan and then snap freeze and perform the cryosectioning.
Does anyone know whether the fixing of the bone or the CT scanning will affect the tetracycling labelling or the cryosectioning process.
Thanks!!

#4 bob1

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Posted 08 September 2011 - 10:20 PM

I have no real idea about the CT but I don't think either will affect either the staining or the sectioning.

#5 blablabla

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Posted 12 September 2011 - 02:02 AM

Thanks very much for your reply, thats good to hear, its the staining that I was most worried about.
Thanks again!




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