Greetings, Below is one protocol suggested for the introduction of antibodies into viable cells that wasn't posted to the entire list: ............................................................................ ............ might try sterile saponin solutions Saponin Method: Permeabilization REAGENT for Paraffin sections & Itracellular Cytokine staining Dulbecco's PBS (without Mg2+ or CA2+) 1% heat inactivated FCS (or BSA-do not use animal serum with Biotin detection systems due to endogenous biotin levels) 0.1% w/v sodium azide 0.1% w/v saponin (Sigma cat#S-7900) Adjust pH buffer to 7.4-7.6 and filter see ref: 1)Sander, B., J. Andersson and U. Andersson, 1991:Assessment of cytokines by immunofluorsecence and the paraformaldehyde-saponin procedure:Immunol Rev 119:65-93 2) Anderson, U. and J. Andersson 1994. Immunolabelling of cytokine producing cells in tissues and suspension. In Cytokine Producing Cells eds. D. Fradelizie and D. Emelie. INSERM, Paris. 32-49. for intracellular cytokines, stop golgi process with with either monesin (Sigma cat#M-5273) or Brefeldin A (Sigma cat#B-7551) -ref#Nature 373:255-257 -Prussin C and Metcalfe DD. Detection of Intracytoplasmic Cytokine Using Flow Cytometry and Directly Conjugated anti-Cytokine Antibodies. Journal of Immunological Methods, 188, 117-128 (1995). -J. Immunol Meth 159:197-207 see antibodies at: http://www.researchd.com/absort.htm Sincerely, Research Diagnostics Inc Pleasant Hill Road Flanders NJ 07836 phone 973-584-7093 fax 973-584-0210 email: researchd@aol.com web: http://www.researchd.com
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