Paraformaldehyde and immunofluorescence - (Mar/19/2009 )
I'm using an Alexa (one of the red ones) secondary antibody. When I fix with 3% paraformaldehyde for 15 minutes and permeablise with 0.025% TX-100 my background is mostly nuclear. I repeated my protocol without the permeabilisation step and it significantly reduced the nuclear background.
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?
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.
bob1 on Mar 20 2009, 12:03 AM said:
I know that. My question is whether some antibody can still enter if I just use paraformaldehyde alone?
No, antibodies are unable to enter the cell without the permiabilization step which explains why your nuclear background staining disappeared. What background you are still visualizing without the permiabilization is most likely antibodies binding the outside of the cell. Try using an antibody that stains a cytoplasmic protein to see it yourself. Without the permiabilization you won't be able to get good staining.
It depends where the protein of interest is localised. If its inside the cell, you need to permebilise it. Try to reduce conc. of secondary ab. may be this helps.
as Scolix said it depends where your protein of interest in localised.
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.
