Posted 11 September 2010 - 02:39 PM
When it comes to IF, I've found that monoclonal versus polyclonal isn't a major consideration. It's more a matter of which antibody works. Polyclonal may give you more background but it's also more likely to stain your specific protein of interest. You should try both if you can. As for controls, you want to test your secondary antibody alone (no primary) to see what background this gives you. You may find you need to mess around with your fixation and blocking methods to optimize but then again, sometimes specific antibodies and cellular structures require a specific fixation. Centrosomes, for example, require methanol fixation. Autofluorescence would be what you see with no antibody staining, which in my experience is next to nothing but may depend on the cells or tissues being analyzed. If you do a google search you'll find ways to deal with or reduce autofluorescence if this is an issue. Generally, aldehyde fixatives are more prone to creating autofluorescence with gluteraldehyde being the worst. The best way to ensure specificity of the primary antibody is to preabsorb the antibody with either the protein of interest or the peptide to which the antibody was generated. Many companies offer the peptide as well as the antibody just for this purpose. You incubate the antibody with the peptide and then try to stain your samples. Any signal you see with this treatment is potentially nonspecific.