BrDU staining on GFP positive cells - (Nov/07/2007 )
Hello all,
Recently I did BrDU staining on stably transfected GFP positive endothelial cells. I fixed cells in MeOH:Acetone (1:1), rinsed in PBS and put aside O/N in PBS at $C. The next day I treated cells in 1.5M Hcl followed by PBS/O.5%BSA fixation for 10 mins, Primary ab was added with DNase and PBS for incubated for 2 hrs follwed by secondary ab and dapi treatment.
My BrDU stained seemed to have worked but shockingly (!!!) I dont see any GFP postive cells. I mean under bright light I can see the cells attached to the plate but cannot detect GFP.
Pls help!!
-ruchi-
It could because fixative is too weak, such that GFP leaked out, or your acid treatment was too harsh to GFP. What is the purpose of HCl treatment?
-genehunter-1-