immunostaining for PML nuclear bodies - Please enlighten me... (Feb/07/2007 )
Could someone please kindly enlighten me here...
I have been trying to do an immunostaining for PML nuclear body in cos7 and Hela cells. However, I saw only faintly stained nucleus and very few nuclear bodies =(
The antibody that i am using is a mouse monoclonal antibody, with TRITC conjugate from Santa Cruz.
After washing the cells wiith PBS, I fixed the cells with pre-chilled 100% methanol for 10min at -20 degree.
After washing the cells in 10%FBS/PBS-0.1%saponin, I incubated the antibody (1:50)with cells on the coverslip for 1hr at RT.
Then I washed the cells with the same washing solution before I stained the cells with DAPI.
The coverslips were then washed with PBS before being mounted to glass slides using Vectorshiefd.
My DAPI staining was very clear under a fluorescence microscope from Ziess. However, the PML nuclear bodies were barely seen in cos7 cells and slightly present in the Hela cells.
Could someone kindly enlighten me what could be the cause(s) of my problem here? Is it necessary to use a confocal microscope to obtain the stained PML nuclear bodies? Almost all published data that I read presented the immunostained PML nuclear bodies from confocal images
Thank you in advance for your help!!!
A troubled ZincFinger
you could try fixing with a 1:1 methanol acetone mix or permeablising the nuclear envelope with 0.2% triton x.
if they dont work you could consider signal amplification (a biotinylated secondary to streptavidin oregon green is pretty bullet proof)
dapi always comes thru strong and i tend to turn down the signal so i dont overload the camera - you could try increasing the signal/sensitivity on your microscope.
and finally - its unlikely you problem lies in your lack of confocal - i'm using a deltavision at the mo which doesnt have a laser on it (filters) but will still find the smallest amount of fluorochrome - when it comes to stuff to attach your fluorochrome to being lacking thats a different story.