[Histonet] processing of whole late stage (E18) mouse embryos(frozen and paraffin)

[Sarah Jones]

Hi Juile,
   The tissue sounds like it is not being dehydrated enough.  What
times are you using on the processor?  What are the dimensions of the
embryos?  What clearing agent are you using on the processor?  What
processor do you have?   

Sarah Jones HT(ASCP)
Dept. of Vet. Anatomy & Public Health
Histology Lab
Texas A&M University
College Station, TX 77843-4458
phone: 979-845-3177
fax:  979-458-3499


>>> Juile Gorenstein <j_gorenstein <@t> yahoo.com> 7/14/2004 2:45:13 PM >>>
Hello everyone,
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,
Julie
 
Julie Gorenstein
Novartis, Cambridge, MA

		
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