Purpose

To describe the steps and reagents needed in the Immunofluorescence Assay (IFA)

References

Localization of Antigen in Tissue Cells (1950), J. Exp. Med., 91:1-13

Materials

  • 1X PBS (from SOP-104)
  • Antigen slide (10 wells per slide)
          # CG015  CMV Antigen Slide OR
          # H1G028  HSV-1 Antigen Slide OR
          # H2G029  HSV-2 Antigen Slide OR
          # VG038    VZV Antigen Slide OR
          # EG039    EBV Antigen Slide
          If making your own slide, fix the cells with acetone or methanol.
  • Primary antibody of interest (human serum/plasma or monoclonal antibody)
  • Secondary antibody labelled with FITC
           # AG002  anti-Mouse IgG-FITC with counterstain OR
           # AG040  anti-Human IgG-FITC with counterstain OR
           # AG041  anti-Human IgM-FITC with counterstain
  • Mounting media (50% Glycerol/1X PBS)
           #1 Coverslip (24 x 50 mm)  VWR 48393-081 or equivalent

Equipment

  • Stir plate and small stir bar (or a metal paper clip)
  • Glassware
  • Humid Chamber (this can be an empty pipette tip box with about 1/2" water in the bottom half of the box).
  • 37°C Incubator
  • Staining dish
  • Pipettors
  • Fluorescence-capable Microscope equipped with Fluorescein excitation filter (490 nm) and a long-pass emission filter (510 nm +)

 

Procedure

  1. Allow slides to warm to room temperature.
  2. 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).
  3. 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.
  4. Incubate the slide in a humid chamber for 30 minutes at 37°C (60 minutes for IgM antibodies).
  5. 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.
  6. Wash in approximately 250 ml PBS in staining dish while stirring for 10 minutes.
  7. 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.
  8. Place 1/2 drop of secondary antibody (see materials section) per well and incubate in a humid chamber for 30 minutes at 37°C.
  9. Rinse off the secondary antibody as before and wash in approximately 250 ml PBS for 10 minutes while stirring in a staining dish.
  10. Remove the slide and air dry.
  11. 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.
  12. Observe in the fluorescence microscope at 400X (10X eyepiece and 40X objective).

Data

Record fluorescent intensities on a scale of 1 - 4 with 4 being very bright and 1 being the lowest intensity that is still obvious.

 

Latest Additions

Your Purchases Today

The cart is empty

OpenWetWare

vipr logo 1