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--------------------------- Original Message ---------------------------
--------------------------- Original Message ---------------------------
Hi all,
I have a basic question on how to fix cells.
We normally isolate lymphocytes and granulocytes from human blood by using
Histopaque centrifugation, and wash with PBS to remove absorbed
Histopaque. These cells are labelled with primary antibodies, and then
with secondary antibodies before flow sorting. We use these sorted cells
for in situ hybridization.
It would be better if we could fix cells before labelling with antibodies.
Fixation of cells with paraformaldehyde requires a proteolytic digestion
step prior to in situ hybridiztion. Therefore, we are avoiding
formaldehyde fixation.
We tried to fix cells with methanol or ethanol (70% to 100%) before
labelling with antibodies. The problem is that cells undergo aggregations
by alcohols. Could somebody suggest how to avoid the aggregation during
fixation or other alternative, gentler fixation methods?
Thanks.
In C. Kim <inkim@carina.unm.edu>
University of New Mexico School of Medicine
Albuquerque, New Mexico
When you fix the cells in EtOH, are you adding the cells to the EtOH
or vice versa. You should always resuspend your pelleted cells
in a small volume of buffer, then pipette rapidly into the cold
(-20C) EtOH. This will avoid most cases of aggregation which
will occur.
======================================================================
Brian Sailer
d9at@sdsumus.sdstate.edu
South Dakota State University
Broookings, SD 57007
In Alaska, if you ain't the lead dog the view never changes!
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======================================================================
Brian Sailer
d9at@sdsumus.sdstate.edu
South Dakota State University
Broookings, SD 57007
In Alaska, if you ain't the lead dog the view never changes!
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