Paraformaldehyde and immunofluorescence
Posted 19 March 2009 - 03:45 PM
I suppose what I would like to know is whether paraformaldehyde on its own can disrupt the cell enough for antibody entry, yet with the permeabilisation step ommitted, the nucleus is resistant to antibody entry?
Posted 19 March 2009 - 04:03 PM
Posted 20 March 2009 - 04:11 AM
If you fix with para, you need to permeabilise the cells before antibodies can efficiently enter the cells. If you have background, make sure that you are washing the paraformaldehyde off properly, at least 4 changes of PBS or TBS will be enough. Leaving overnight at 4 deg C in PBS helps too.
I know that. My question is whether some antibody can still enter if I just use paraformaldehyde alone?
Posted 20 March 2009 - 07:08 AM
Posted 21 March 2009 - 10:34 PM
Posted 29 March 2009 - 01:46 AM
but personally I really don't think permeabilization with Triton x100 is necessary in case of DAPI and Hoechst staining, because we do this every day in our lab and we never permeabilize. we stain less than 1 minute with 1ug/ml of DAPI or Hoechst after fixation with 3% Paraformaldehyde.
I've even detected proteins in cytoplasm and mitochondria membrane without permeabilization.
however I've never used antibodies that target nuclei proteins. maybe you would need to permeablize in case your protein of interest is inside nuclei.