CIHRT Exhibit P-3666 Page 1 Pathology Division Section: Anatomic Pathology / Technical Procedures Number: PRC-PAT-(IHC)- Title: Direct Immunofluorescence Procedure Page 1 of 3 Issuing Authority (s): B.Dyer, Pathology Manager Author: B.Rowe Signature: Date: Issue Date: Date Effective: Revision Dates:

Purpose

This procedure provides instructions to perform direct immunofluorescence (DIF).

Materials Supplies Equipment

Coverslips • Antibodies • Cryostat Diamond • PBS buffer or Ventana • 37 degree oven tip marker Reaction buffer. • Humidity chamber Coplin jar • Fluorescent mounting • Microscope medium • Acetone

Special safety precautions

Use personal protective equipment including gloves, goggles and chemical resistant aprons.

NOTE: Perform DIF procedure with minimal amount of light exposure.

Procedure

Follow the activities below to perform direct immunofluorescence. Please read all instructions before starting the procedure.

1. Cut frozen section slide at 5um. 2. Stain slide with Toluidine Blue. If the tissue is kidney view slide to check for glomeruli. Do not proceed with DIF if no glomerulus is seen. If the tissue is skin proceed with DIF if the epidermal layer is present. If the DIF is not done, the pathologist is to be informed and a comment must be placed under SPEC COMMENTS in Meditech. 3. Cut twice as many slides as needed per protocol. 4. Label slides with surgical number and antibody identification. 5. Etch a circle around the tissue on the glass slide with a diamond tip marker. 6. Place slides in acetone for 5 minutes. CIHRT Exhibit P-3666 Page 2 Pathology Division Section: Anatomic Pathology / Technical Procedures Number: PRC-PAT-(IHC)- Title: Direct Immunofluorescence Procedure Page 2 of 3 Issuing Authority (s): B.Dyer, Pathology Manager Author: B.Rowe Signature: Date: Issue Date: Date Effective: Revision Dates:

7. Remove from acetone and allow to air dry for 5 minutes. 8. Place slides in PBS (Ventana reaction buffer) for 5 minutes. 9. Prepare direct antibody dilutions during this time. All dilutions must be prepared with PBS (Ventana reaction buffer) as follows: Skin Biopsies Protocol Renal Biopsies Protocol IgG 1/100 IgG 1/100 IgA 1/40 IgA 1/40 IgM 1/40 IgM 1/40 C3Comp 1/100 C3Comp 1/100 C1Q 16.6/100 Kappa 1/40 Lambda 1/40 Fibrinogen 1/100 Albumin 1/40 10. Put a minimum of 200 mls. of water in the humidity chamber. 11. Place slides in humidity chamber Pipette 100ul of diluted direct antibody to appropriate slide within the etched area. 12. Cover the chamber and place in the 37 degree oven for 30 minutes. 13. Remove chamber and cover; wash well three times with PBS (Ventana reaction buffer). 14. Drain slides, coverslip with Dakocytomation Fluorescent mounting medium. 15. Place requisition in slide folder with the stained slides then place in IHC refrigerator. 16. Store unstained slides in -70˚ freezer.

References

[LIST REFERENCES HERE, AS APPROPRIATE]

Related procedures Transportin IF stained slides

CIHRT Exhibit P-3666 Page 3 Pathology Division Section: Anatomic Pathology / Technical Procedures Number: PRC-PAT-(IHC)- Title: Direct Immunofluorescence Procedure Page 3 of 3 Issuing Authority (s): B.Dyer, Pathology Manager Author: B.Rowe Signature: Date: Issue Date: Date Effective: Revision Dates:

[LIST RELATED PROCEDURE(S) HERE, AS APPROPRIATE]

Documents/Forms/Records

[LIST REQUIRED DOCUMENTS, FORMS, AND RECORDS HERE, AS APPROPRIATE]