Troubleshooting Immunofluorescence Assays
Problem: Positive Control is negative or very weak. Possible Causes:
1. Please note that Controls available from Fuller Laboratories have been bottled at “screening dilution”. This dilution depends upon the specific assay, but in all cases Positive Controls are intended to be used initially without dilution to demonstrate a “strong” reaction. These Controls give endpoint titers 8-fold beyond the screening dilution (range 1:4-1:16).
2. If IFA Substrate Slides are purchased as components, the Positive and Negative Controls available from Fuller Laboratories may be used to locate the optimal conjugate titer for use. This optimal titer should give a clear 1+ positive reaction at a Positive Control dilution of 1:8 (see #1 above) and a clearly negative reaction using the undiluted Negative Control.
3. Microscope must be equipped with a filter system for AlexaFluor 488 (maximum excitation wavelength 493 nm, mean emission wavelength 518 nm) and 400X magnification.