Triton not permeabilizing nuclear envelope - (Dec/16/2005 )
Hello, I am hoping someone can help me:
When I fix my primary cultured cancer cells in 4% PFA (20min) and then permeabilized in 0.5% Triton (10min) instead of getting nice nuclear fluorescence of specific structures, I get just nuclear envelope staining and almost no internal nuclear staining (but DAPI works perfectly). If I instead do 4% PFA with 0.5% Triton together (20 min) then the staining works fine but the cells are smaller and the structures are pushed together. I would like to get this procedure working (it seems to be what most people use) but so far have been unsuccessful. It happens with both polyclonal and monoclonal primary and secondary antibodies. I have tried everything I can think of:
Varying Triton concentration from 0.05% up to 5%
Trying 3X5min incubations of 0.5% Triton
Having 0.5% triton in with the antibodies (after already incubating with Triton on its own)
Varying incubation of 0.5% Triton time from 5 min up to 30 min
If anyone has a suggestion as to a possibility why the secondary (or possibly the primary) can't get into the nucleus after the Triton permeabilization after fixation instead of during, I am open to any suggestions.
I know someone who permeabilizes with cold methnol for 10 mins after PFA fixing...
Have you tried Triton + ammoniumchloride???
you could fix in 4% paraformaldehyde before using triton-x before your blocking step
I commonly did confocal on cancer cell lines looking at nuclear localisation using 4%PFA fixative with no discernable difference in morphology. To permeabilise the cell I use 0.3%Tx100/0.025% CHAPS in PBS for 20 min at room temp. The CHAPS is added to permeabilise the nucleus.
Hope this helps