I have performed whole mount immunos on day-3 chick embryos and used a fluorescent secondary antibody. I would now like to process the tissue for sectioning and was wondering whether the best technique is cryosectioning or whether there was a way to embed the tissue into paraffin?
I have done whole mount ISH stains, which someone else then cut in a cryotome. The hardest part was orienting the fish embryos in the freezing medium...
I doubt your fluorescent stain will survive paraffin processing.
Thanks Jonathan. I think you are right. The xylene and paraffin will probably do something to the fluorescence.
I agree, xylene will remove fluorescence, but I would suggest to look on embryos without any sectioning. If you make enough good permeabilization step, you can achieve good antibody penetration even to inside layers. I am not sure you can make a good images from internal part of the embryos without sectioning, but you should have enough good signal in the outer layers. I hope this will help.
Actually, sectioning should be done before antibody incubation.
I suggest you use glycol-methacrylate resin (like Historesin, Leica) for embedding your material after immunofluorescence step. I did it and worked great.
Embed in agarose, or (gelattine-albumine) : do vibratome sections
I agree with Irina, the best approach is to embed in a low-melting point agarose and use a vibratome, which you should be able to get sections ~60um thick. Because of the minimal tissue preparation required, you should not lose any of your fluorescent signal and can complete with the tissue in the dark. Using the agarose, will also enable you to orientate the tissue with a high level of accuracy. Another great advantage to this approach, is that most histology facilities have such equipment and as long as the equipment has been maintained, is very precise, even if old.
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