Re: ISH for mRNA

From:Tracey Couse

Bob,

For all of our ISH work we used 4% paraformaldehyde including fixation of 
our cell monolayers.  For our monolayers grown on chamber wells, we would 
rinse in PBS or Hank's balanced salt solution then fix in fresh 4% 
paraformaldehyde for 10 minutes, rinse again and then store in 70% alcohol 
until we were ready to do the experiment.  I am unclear on how you are 
growing your cells and how you will prepare them for paraffin processing, 
but to answer your question, the time of fixation will depend on the 
thickness of your cell preparations.  If you are fixing a 'tiny' (how 
ambiguous is that?!?) cell pellet then fix for at least 1-2 hours (remember 
formaldehyde fixation rate is about 1mm per 24 hours at room 
temperature).  Hope this helps.

Tracey Couse
Petit Institute for Bioengineering & Bioscience
Georgia Institute of Technology


Date: 18 Oct 2001 17:19:37 -0500
From: r-meyer2@northwestern.edu

Subject: ISH for mRNA
Does anyone know if 4% Paraformaldeyhde is the best fixative for cultured
cells
that will be paraffin embedded for in situ hybridization for mRNA? If so what
is the time of fixation for these cells? I'm thinking probably 1 to 2 hours
is
plenty. If 4% Paraformaldehyde isn't the best fixative does anyone know which
fixative will give the best results. Thanks
Bob Meyer, HTL
Northwestern University 





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