[Histonet] Double staining of 2 antibodies with 2 different retrieval methods

[Patrick Laurie]

Hello Histonet,

 

I am trying to do some double labeling of ubiquitin and HLA-DR LN3
antibody on human spinal cord. My PI wants to be able to see both the
ubiquitinization of the motor neurons with patients that had ALS, as
well as seeing the proliferation and activation of microglia on the same
slide, near the same cell.  But, I have found that the ubiquitin I am
using, (Chemicon's Monoclonal ubiquitin antibody) requires no antigen
retrieval (AR), while the HLA-DR LN3 antibody (monoclonal from MP
biomedical) works best with a 30 min citrate retrieval.  The citrate
retrieval with the Ubiquitin creates too much background staining,
blocking reagents are powerless to stop it.  Sooo, to make a long story
short, what would be the easiest way to get my PI's (not my) desired
result?  He wants to stay with chemicons monoclonal ubiquitin, Dako's is
a polyclonal, but it also doesn't want AR.  Does anyone have any
suggestions, is this a hopeless cause?

 

Thanks in advance for your advice,

 

Patrick Laurie, HT (ASCP)
Neurogenomics Laboratory
Benaroya Research Institute
1201 9th Ave
Seattle, Wa  98101
(206) 341-0681