Helpful Hints for Using Exalpha Immunofluorescence Kits
- 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.
- To help decrease autofluorescence, add a Sudan Black step after the SA-FL incubation.
- Make up the Sudan Black at 0.2% in 70% Ethanol (mix for 2 hours in the dark).
- After the SA-FL incubation and wash step, add 100 µL of 0.2% Sudan Black to the slide.
- Incubate for 15 minutes at room temperature in the dark.
- Wash 8 X 1 minute using the wash buffer recommended in the assay specific protocol.
- Mount and view the slide under the microscope.
- While the use of Sudan Black can help decrease autofluorescence, it can also decrease signal. Sudan Black should be used with caution.
- The addition of 0.02% tween to the wash step following the Sudan Black incubation may help to increase the signal.
- Sudan Black should be made up fresh each time.
- Sudan Black can be tested at at a lower concentration (e.g., 0.025%).
- Completed slides should be stored in the dark when they are not in use.
- Slides should be analyzed shortly after the mounting media is dry.
The signal is
difficult to find
- 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.
- Make sure that the work area surrounding the microscope is dark.
- Allow your eyes to adjust to the dark prior to viewing the slides.