Intracellular staining of eosinophils

Dawn Cooper dlc32 at hermes.cam.ac.uk
Wed Feb 2 05:26:03 EST 2000




I am wanting to measure activated eosinophils with the monoclonal
antibodies EG1 and EG2 and have identified a method from previous
published papers , the FOG method, and wondered if any one would have any
experience  with this technique?

the FOG method should permit the detection of intracellular antigens in
unseparated peripheral blood by flow cytometry. This uses paraformaldehyde
to fix the leukocytes and the detergent
n-octyl-beta-D-glycopyranoside(OG) to permeabilise them.
I am then staining with monoclonal antibodies specific for eosinophil
cationic protein EG1 and EG2 and a second FITC-conjugated mouse IgG.

However i am getting staining of Lymphocytes as well as eosinophils. I
wondered if anyone had any experience with this technique or problem
before, or could suggest steps in the method which could be causing this
problem if done wrong as i have little experience in this area?

Many Thanks
Dawn Cooper


Dawn Cooper
Department of Clinical Biochemistry
Box 232
Addenbrooke's Hospital 
Hills Road
Cambridge
CB2 2QQ
England

tel: 01223 217337





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