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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.

2015 Steering Committee Meeting

The next EDRN Steering Committee Meeting will take place March 31st through April 2nd, 2015, in Atlanta, Georgia.

Announcement 12/02/2014

New Round of EDRN FOAs

The RFAs for EDRN have been released:
- Biomarker Developmental Laboratories (U01),
- Clinical Validation Centers (U01),
- Biomarker Reference Laboratories (U24),
- Data Management and Coordinating Center (U24).

EDRN Renewal flyer NOTE-New receipt deadline for applications submitted for all EDRN FOAs is January 20, 2015, by 5:00 PM local time of applicant organization.

There will be a Pre-Application webinar to discuss each of the four individual EDRN FOAs on Tuesday, December 2nd, 2014, from 1pm-5pm (Eastern). Potential applicants interested in participating in the webinar should send a message to Dr. Sharmistha Ghosh (ghoshjanjigias@mail.nih.gov) no later than 5:00 p.m. (EST) November 21, 2014. Please mention the FOA of interest in the subject line.

Announcement 10/07/2014

EDRN Patient Advocates will host an EDRN Advocacy Educational Webinar, Biomarkers for Prostate Cancer Detection and Monitoring, on Monday, January 12th, 2015, at 1 p.m. EDT / 10 a.m. PDT. Registration is not required for this. Please click for more information.