[Histonet] immunohistochemistry and TUNEL background staining

[Jacqui Detmar]

Hi all.  I have been doing IHC and TUNELs on mouse placental tissues for the past 4 years and have been having problems with oodles of background staining during the past 6 months or so.  I have tried doing avidin-biotin and streptavidin-biotin blocking and also using ABC (purchased from Vector) in high pH (TBS, pH 9.4) or high salt (TBS [NaCl = 0.5M], pH 7.6) solutions.  Nothing seems to be working.  The IHC using biotinylated secondary antibodies and the TUNEL is done using biotinylated dUTP.  I have tried using old sections that I *know* have worked in the past and I am getting the same type of background staining as my newer blocks (i.e. the problem is likely not due to fixation (always the same...24 hours in 10% phosphate-buffered formalin), nor due to differences in paraffin embedding).  

So, could my xylene and ethanol solutions be a problem?  We get the ethanol in-house here at the hospital and the Xylene is purchased through Fisher (X16-4).  What about differences in TBS or PBS (I have tried using both to see if the problem would go away and it doesn't make a difference).  

Any advice would be appreciated.

Regards,

Jacqui Detmar, Ph.D. student
Samuel Lunenfeld Research Institute
Mount Sinai Hospital,
Toronto, Ontario, Canada