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You are here: Home / Resources / EDRN Standard Operating Procedures (SOP) / Assays / Immunohistochemistry / Immunofluorescence Staining

Immunofluorescence Staining

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

Specimen

Frozen tissue only.

  1. Fresh tissue (kidney, lung, skin, etc.) is frozen in -80°C freezer.
  2. Embedding: OCT medium sectioning at 7μm.

Controls

  1. Use a frozen control of tonsils.

Procedure

  1. Place some OCT in plastic freezing boat.
  2. Place tissue in OCT and orient according to standard embedding protocol.
  3. Freeze in -80° Freezer.
  4. Remove block from freezer and place in cryostat.
  5. Cut sections from frozen block and place on coated slides. Sections should be cut at 7 microns on the cryostat.
  6. Fix in cold acetone for 10 minutes.
  7. Air dry.
  8. Place slides in washing PBS for 5 minutes. OCT medium will dissolve in this wash.
  9. Place antibodies on slides for 30 minutes. Slides must be kept in dark.
  10. Rinse in PBS.
  11. Coverslip with fluorescent mounting medium.
  12. Store slides in cardboard folder in refrigerator until ready to screen.

Note: Using the Ventana automated procedure, slides will be placed on machine at Step 9 and resumed with Step. 10.

Announcement 10/23/2017

Three new FOAs on the Human Tumor Atlas and associated with the Cancer Moonshot Initiative have been released. Please click here for more information.

10th Science Workshop
Thank you to everyone who made the 32nd EDRN Steering Committee meeting a success. The next event is the 10th EDRN Scientific Workshop from March 6-8, 2018 in Bethesda, MD. Click to view the flyer; click to visit the registration page.
EDRN Founder Honored

Dr. Sudhir Srivastava was honored with the Distinguished Service Award from the American Pancreatic Association at the group's annual meeting this year, for his outstanding commitment to pancreatology.