[Histonet] non specific staining

[Kim O'Sullivan]

Hi,

I am having a problem using DAB black on snap frozen mouse kidneys. We routinely stain for leukocytes (CD4(Gk1.5),neutrophils(GR1), and macrophages (fA11)) on PLP fixed tissue with very little background staining. However we currrently have to stain some snap tissue as the PLP tissue is all gone. 

The problem we are having is some cells are staining very specifically on the isotype controls, and controls with no primary or secondary antibody-this population of cells are hard to distinguish from positive cells in the experimental tissue.

I presume it is the DAB chromagen that is binding very specifically to a certain cell population. We currently use 0.3% H202 in methanol for 30 minutes to block endogenous peroxidase- is this insufficient? Does anyone think it is an endogenous peroxidase problem or something else?

Any suggestions would be helpful,

Kim
Department Of Medicine
Monash University
Monash Medical Centre
Melbourne, Australia