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Troubleshooting Immunofluorescence Assays

Problem: Positive Control is negative or very weak
Possible Causes: 
1.
Microscope must be equipped with a filter system for FITC (maximum excitation wavelength 490 nm, mean emission wavelength 530 nm) and 400X magnification.
Problem: Antigen or substrate cells smeared or absent from wells before test
Possible Causes: 
1.
Substrate slides must be allowed to equilibrate with ambient temperature prior to being opened. Opening slide packets with slides still cool will quickly lead to moisture condensation and produce an antigen suspension in the condensate liquid. Such slides are not useable. Allow at least 15-20 minutes for temperature equilibration, with the slides individually laying on the bench surface (not stacked).
2.
Substrate slides are routinely checked for vacuum leakage prior to shipping. If you have received slide packets that are not flat beyond the edges of the substrate slide, please notify your supplier.
Problem: Antigen or substrate cells washed off during the procedure
Possible Causes: 
1.
It is possible to wash a substrate slide too much. EnoGene recommend, especially for IgG assays, that the slides be gently washed using a Wash Bottle with PBS. Aim this 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 add the conjugate. For IgM assays it is recommended that the wash buffer (after the third wash) be allowed to remain inthe 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 been scratched off by the technician in applying sera or conjugate. Application of sera or conjugate should be done from the edge of the substrate wells.
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.
Problem: Reactions are stronger on the edges of the well in comparison to the center.
Possible Causes: 
1.
This phenomenon depends upon cell distribution within each well and the effects of the liquid droplet. As the edge of the well generally has less antigen, reactivity may appear increased due to an increase in the antibody-to-antigen ratio. This effect is amplified by the kinetic energy associated with surface tension near the edge of the serum droplet. Assays are standardized using reactivity in the central portion of the well and normal ranges are based upon this reading.