Troubleshooting Immunofluorescence Assays
Problem: Antigen washed off during the procedure.   Possible Causes:
1. It is possible to wash a substrate slide too much. We recommend, especially for IgG assays, that the slides be gently washed using a Wash Bottle containing PBS. Aim the stream of wash buffer to gently strike the middle of the slide, not the antigen wells. Do not immerse in wash buffer for long periods or douse with distilled water, simply use the wash bottle 3 times and apply the conjugate. For IgM assays it is recommended that the wash buffer (after the third wash) be allowed to remain on the slide wells for several minutes before removal and addition of conjugate.
2. If only some of the sera tend to remove the substrate, the problem may be bacterial proteases due to bacterial growth in the sera.
3. If the loss of substrate is in patches or streaks, the substrate has possibly been scratched off by the technician in applying sera or conjugate while performing the assay. Application of sera or conjugate should be done from the edge of the substrate wells.