immunostaining COS-7 cells

Nancy David ndavid at bcm.tmc.edu
Fri Nov 4 13:23:48 EST 1994


In article <ciiYtia00iV442TUcD at andrew.cmu.edu>, "Jeffrey D. Bray"
<jbdq+ at andrew.cmu.edu> wrote:

> Hi,
> 
> I wish to transfect COS-7 cells with 2 different expression vector
> plasmids containing the tropomyosin and NM/SM myosin light chain tagged
> with the HA-epitope. I would then like to perform immunofluorescence to
> see if there is correct localization of these gene products.  Does
> anyone have a good protocol for immunostaining COS-7's or stress fiber
> proteins.  Thanks in advance.
> 
> Jeff Bray CMU grad student <jbdq+ at andrew.cmu.edu> 

-- 
I did some nice immunostaining of cos-7. 
1. Fix in paraform. 7 min
2. Triton X100 5 min
3. Heat inactivated goat serum 15 min
4. Primary Ab 1h or more (even overnight) (need just cover the whole
coverslip)
5. Wash 4 times (5min each)
6. Secondary Ab 30 min or so
7. Idem 5
8. Mount in mowiol 
9. Go to scope.

Usually I do two fast washes in filtered PBS between each step. Fiklter all

solutions (better background). Steps 1, 2 and washes I do in six wells
plate, 
the rest on the lid of a petri dish covered with parafilm. After step 3
don't
wash, just blot the serum off with a kimwipe.
As secondary Ab, I believe that Fab fragments work better, but don,t take
that 
seriously. 
If you need some more info (details, etc) email me or post again.

Ramiro Salas 
Baylor Col of Med, Houston TX
email rs690935 at mbcr.bcm.tmc.edu



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