Protocol Online logo
Top : Forum Archives: : Immunology and Histology

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!!


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?