My staining works fine with purified followed by a secondary antibody, but the eFluor® Nanocrystal conjugated antibody does not appear to be working. What could be wrong?
Although the protocol works for two-step or three-step staining, the conditions may need to be changed for staining with eFluor® Nanocrystal conjugates. Here are several points to consider: 1. Protocol- We recommend using the protocols developed by eBioscience that have been shown to work for a variety of direct eFluor® Nanocrystal conjugated antibodies. Please refer to our recommended protocols in the BestProtocols section of our website. 2. Blocking buffer- We have formulated two versions: one containing high amounts of protein and another with lower levels. We recommend starting with IHC/ICC Blocking buffer-Low Protein (Cat. No. 00-4953), which has been shown to be optimal for general use when staining cytoskeletal, cytoplasmic and membrane proteins. For improved specificity and better signal-to-noise ratio, the IHC/ICC Blocking Buffer- High Protein (Cat. No. 00-4952) has been found to give superior results especially with antibodies to nuclear antigens and FFPE tissue. 3. Incubatio
