Problems with BrdU staining - (Jan/13/2006 )

Hi!

First post...Hope i am getting the right form...

I have a problem with BrdU staining with the BrdU staining kit from roche on sympathetic neuronal cultures.
If i follow exactly the roche provided protocol i get 100%!! brdu positive cells and cytosolic staining with a 3h brdu pulse. i then turned to a workgroup intern protocol for brdu staining with a denaturation step with 2N HCl and washing with 0,1M borate buffer ph 8,5.about 10% are stained now with heavy reduced cytosolic staining.

Anyone has an idea why i get 100% positive cells with the manufacturer protocol? Should be impossible in this time frame. Some DNA reconstruction procedures?Fragments that are killed with 2N HCl denaturation? Is the 10% experiment reliable then?

Flourescent staining is over a-mouse biotin (1:200) and streptavidin cy3 (1:100)...perhaps alexa test?

Hope anyone had this problem before
Tobi

Good news! The 10% Experiment seemed to be reliable proven in more than 10 other experiments!

Now i have a new problem! This BrdU protocol needs Denaturation. Now i want to monitor GFP-transfected cells too. The HCl seems to denature the endogeneous GFP too. Is there a more acid resistable GFP? Perhaps an anti-GFP antibody? Or may i choose another method to denaturize the DNA for the anti-BrdU? Maybe temp.?Or stain for myc-tagged GFP?!

Thanx for suggestions
Tobi

Nobody has an idea?

just use Brdu STAINING KIT FROM Zymed. (CAT#93-3943). It works perfect.