Simon, If your unconjugated antibody cannot block the staining of the conjugated version (assuming you are blocking with a decent excess of unlabelled reagent), your problem may lie elsewhere. I assume you are using a FITC conjugate. If not, the following may not apply. Most vendors of FITC conjugates market reagents with a high F:P ratio of about 6; ie there are 6 FITC molecules on each antibody. While this situation is generally good for surface staining, it is generally no good for cytoplasmic staining. For the latter, F:P ratios of about 1 are optimal. Since FITC is a negatively charged fluorochrome, normal FITC conjugates acquire extra net negative charge, and it thought that electrostatic interractions of such negatively charged conjugates with cytoplasmic elements cause the problem you see. Cold antibody will not block this 'non-specific' staining. In the old days, we used to perform ion exchange chromatography on such conjugates to separate the conjugates with low F:P ratios. This is a technically painful and relatively little used (these days) method of obviating this problem. One relativley quick and dirty way to 'clean-up' a test batch of your conjugate for cytoplasmic use, is to adsorb it with pig liver powder. Buy this from Sigma or similar. It is a freeze-dried powder, that needs to be washed four or five times with PBS prior to addmixing with your conjugate for 30 minutes or so in the cold. Then remove the solids by centrifugation in a microfuge, and see if it now works. If your problem was as described above, your non-specific staining levels should be lower or gone altogether. Note, you may need to do this trick more than once if you need to use the adsorbed conjugate at the higher concentrations necessary post adsorption. You may want to try other clones too. Not all monoclonal conjugates will be problematic. I suspect that most vendors' isotype controls have quite low F:P ratios, or have been deliberately selected/conjugated/diluted to stain essentially nothing at all!! People say that you should also use isotype conjugates from the same company that supplied your other reagent because different companies use different chemistry, sources of fluorochrome etc in the manufacture of their conjugates. For the above reasons and many others, we and an increasing number of others no longer routinely use them for surface staining studies [see Keeney et al: Perspectives: Isotype controls in the analysis of lymphocytes and CD34+ stem/progenitor cells by flow cytometry - Time to let go! Cytometry (Comm in Clin Cytometry) 34: 280-283, 1998]. I hope this is helpful. cheers, D. Robert Sutherland, Associate Professor, Dept. Medicine, University of Toronto, Canada.
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:31 EST