nuclear counter staining - TOPRO-3 (Oct/08/2007 )
Is anybody using TOPRO-3 from Molecular probes for nuclear counter staining in Immunocytochemistry?
I am using 1uM (1:1000 dilution in PBS) for 30min at RT but the staining is not nuclear specific, I get also cytoplasmic staining.
I think this cytoplasmic staining is because TOPRO is not DNA specific and stains also RNA.
I am planing to do a RNAse treatment in order to degrade RNA and reduce cytoplasmic unspecific staining.
Can anybody advise me how to proceed for this RNAse A treatment?
i've never heard of topro 3
if however your immuno is fluorescant toto-3-iodide (633 laser) or dapi (in the vectashield 350 i think) are both reliably nuclear
if not try a standard histology book, haematoxylin might be a place to start (might be too dark) or a weak gram stain (without the cytoplasm bit - its been a while so i cant remember which that is)
you might consider modified harris haematoxylin soln (sigma) - if its not fluor of course
You should use a higher concentration of topro. I have read dilutions in PBS about 1:100, because the dye leaves the cells quickly