[Histonet] processing of whole late stage (E18) mouse embryos (frozen and paraffin)
I have been trying to process and section late stage embryos both for paraffin and frozen sections. I would like to be able to do histology, ISH and IHC on paraffin sections, as well as LacZ staining on frozen sections (saggital).
I have heard that I need to fix in PFA (NBF) to be able to do ISH and IHC. Is this necessary? Has anyone ever tried to do ISH and IHC on Bouin's or Carnoy's fixed tissue? Could anyone suggest what is the best way to fix these guys without mutilation (ie separation of head from the body or making large incisions)?
I also had problems dehydrating the tissue prior to paraffin embedding, even using tissue processor. I think it's dehydration step, anyways. The tissue comes out soft and whitish in places. I have tried to reprocess that tissue (manually and with a processor deparaffinize, then reprocess again using the tissue processor). This makes the tissue overly dehydrated and crumbly when attempted to section. Could anyone suggest an appropriate program to use the first time?
Also, I have been having problems cutting frozen sections (saggital of whole E18). The tissue separates from OCT, and some of the tissues crumbles, depending on temperatures I use on the cryostat. I have tried to use -10oC sample temp, but then OCT has problems being cut. Any suggestions? Is there anything I can do to make OCT stick better to the skin? Are there temperatures that most fit for various types of tissues to be attempted to cut at once?
I am sorry this request has become so large. Any suggestions will be really appreciated.
Thank you all,
Novartis, Cambridge, MA
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