Table 1.
Protocol for immunofluorescence on paraffin embedded renal biopsies
| Cut formalin fixed paraffin embedded tissue at 3-4 μ thickness on poly-L-Lysine coated slides |
| Deparrafinize and rehydrate tissue sections |
| Immerse in Tris EDTA pH 9 for 30 min at room temperature |
| Perform enzymatic digestion with proteinase K 1.25 mg/mL (Sigma Aldrich, United States) at room temperature for 15 min1 |
| Stop digestion by immersing in Tris EDTA at 4 °C |
| Leave in Tris EDTA for 40 min at 4 °C |
| Rinse in PBS for 10 min |
| Apply FITC conjugated polyclonal rabbit antibodies directed against IgG (dilution 1:50), IgM (1:60), IgA (1:60), C3 (1:30), C1q (1:30), kappa (1:25), and lambda (1:40) (BIOSB, Santa Barbara, CA, United States). Incubate for 2 h in a moist chamber in the dark |
| Rinse with PBS |
| Mount in glycerine |
| Examine slides under a dark field immunofluorescence microscope |
1
Varied based on room temperature. PBS: Phosphate buffered saline; FITC: Fluorescein isothiocyanate.