To describe the steps and reagents needed in the Immunofluorescence Assay (IFA)
Localization of Antigen in Tissue Cells (1950), J. Exp. Med., 91:1-13
- Allow slides to warm to room temperature.
- Dilute human serum/plasma 1:20 in PBS or if using monoclonal antibodies dilute appropriately in PBS (use Certificate of Analysis titration data to determine appropriate dilution).
- Apply 15 µl diluted antibody sample per well of the antigen slide. Apply 15 µl PBS to one well to act as the conjugate blank. Take care to spread the drop evenly without letting the pipette tip touch the cells that are on the glass.
- Incubate the slide in a humid chamber for 30 minutes at 37°C (60 minutes for IgM antibodies).
- Gently rinse off the antibody samples with a stream of PBS from a squirt bottle. Take care not to directly hit the cells on the glass.
- Wash in approximately 250 ml PBS in staining dish while stirring for 10 minutes.
- Remove slide and air dry. This can be accelerated by placing the slide on the plenum of a laminar flow biologic safety cabinet or by using a hair dryer on the low heat setting - take care to keep the dryer far enough away from the slide so that the air is not hot when it hits the slide.
- Place 1/2 drop of secondary antibody (see materials section) per well and incubate in a humid chamber for 30 minutes at 37°C.
- Rinse off the secondary antibody as before and wash in approximately 250 ml PBS for 10 minutes while stirring in a staining dish.
- Remove the slide and air dry.
- Apply 1/4 drop 50% Glycerol/PBS per well and carefully apply the coverslip so as not to trap any bubbles. Wick out excess mounting solution by turning the slide on edge on top of a paper towel.
- Observe in the fluorescence microscope at 400X (10X eyepiece and 40X objective).
Record fluorescent intensities on a scale of 1 - 4 with 4 being very bright and 1 being the lowest intensity that is still obvious.