Helpful Hints for Using Exalpha Immunofluorescence Kits

Issue & Tips

Autofluorescence

1. One method of checking for autofluorescence is to look at the slide with multiple filters. If the same signal is seen with multiple filters, it could be autofluorescence.
2. To help decrease autofluorescence, add a Sudan Black step after the SA-FL incubation.
   1. Make up the Sudan Black at 0.2% in 70% Ethanol (mix for 2 hours in the dark).
   2. After the SA-FL incubation and wash step, add 100 µL of 0.2% Sudan Black to the slide.
   3. Incubate for 15 minutes at room temperature in the dark.
   4. Wash 8 X 1 minute using the wash buffer recommended in the assay specific protocol.
   5. Mount and view the slide under the microscope.

Suppressed Signal

If Sudan Black was used:

1. While the use of Sudan Black can help decrease autofluorescence, it can also decrease signal. Sudan Black should be used with caution.
2. The addition of 0.02% tween to the wash step following the Sudan Black incubation may help to increase the signal.
3. Sudan Black should be made up fresh each time.
4. Sudan Black can be tested at a lower concentration (e.g., 0.025%).

Additional tips:

1. Completed slides should be stored in the dark when they are not in use.
2. Slides should be analyzed shortly after the mounting media is dry.

The signal is difficult to find

1. Use the bright field setting on the microscope to focus in on the correct section of the tissue prior to using the excitation wavelength listed in the kit specific protocol.
2. Make sure that the work area surrounding the microscope is dark.
3. Allow your eyes to adjust to the dark prior to viewing the slides.