[Histonet] RE: Cell permeabilization and cell surface staining

[C.M. van der Loos]

   Dear Sonya,

   Sometimes  theory  and  practice doesn't match with each other at all!
   That   keeps  us  awake  and  makes  our  life  both  frustrating  and
   interesting!  I  am afraid that the situations as you sketched are not
   that  black-and-white in real life. For example there is a good change
   that  the  cells  got a bit damaged, allowing the antibodies go in and
   out  and  having  your internal proteins nicely stained. We once saw a
   difference  between  a  cytospin and cells that were attached to glass
   (using  the good old BioRad slides). The attachment procedure appeared
   to  be  more  gentle to the cells than spinning them down with a great
   force, causing damaged cells at least at molecular level.

   Furthermore there might be a molecular reason why the internal antigen
   stained without Triton X100 treatment. Recently, Dick Dapson showed in
   JOH that  formalin  fixation  followed by alcohol dehydration may even
   flip  certain  macromolecules  inside-out.  If  this happens with your
   'internal'  protein you will get nice staining without the Triton X100
   treatment.

   Why  the  Triton  X100  treatment  decreased  your surface staining is
   difficult to understand. First thing that came up with me is that your
   antigen may shed from the membrane due to the Triton X100 treatment.

   Hope this helps a bit.

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands


   Date: Thu, 30 Nov 2006 10:27:57 -0000
   From: "Martin S." <sonya.martin <@t> soton.ac.uk>
   Subject: [Histonet] Cell permeabilization and cell surface staining
   To: <histonet <@t> lists.utsouthwestern.edu>
   Hi all,

   I  have  been staining BMDC's to look at proteins which are present on
   the
   cell surface and in the cytoplasm. I fix the cells with 4%
   paraformaldehyde  in  PBS,  20min  on  ice  and permeabilize with 0.2%
   Triton
   X100 in PBS for 20min at room temp. My two problems are:

   1) When I dont permeabilize the cells I can still detect internal
   antigen  with  my primary antibody (cells are fixed 4% PFA, washed PBS
   and
   blocked with 2% BSA before incubation with antibody)

   2) When I permeabilize the cells I get a decrease in the cell surface
   staining

   Any suggestions greatly appreciated.

   BW
   Sonya