Troubleshooting Immunofluorescence Assays
Problem:¬†Negative Control is positive.   Possible Causes:
1. When serum specimens are initially washed from the substrate wells, the gentle stream of PBS should be aimed to strike the middle of the slide (the mask) to remove the sera from top and bottom rows directly into a waste container or sink. Diluted sera washed across a neighboring well can influence the reaction in that well. This is especially true for high titered sera surrounded by wells containing negative sera.
2. Another possible cause is similar in nature, an inadvertantly sharp movement of the slide or incubation chamber mixes samples from well to well. Careful inspection of the slides after incubation may note different well volumes or liquid on the slide mask between wells.