Abstract:

BACKGROUND: Formalin fixed paraffin embedded tissue (FFPE) is apt for storage, and immunofluorescence method supports precise localization and quantitation as well as multiple labeling.
OBJECTIVE: To establish an upgraded immunofluorescence method for FFPE tissue.
METHODS: Paraffin sections of synovial vascular mantle and acetabular hyperplasia were obtained from rheumatoid arthritis and osteoarthritis patients and then preformed with routine dewaxing and rehydration. The retrieve durations, serum blockage time and primary antibody concentrations of different citric acid buffer and the color difference of fluorescent markers in the immunofluorescence were observed.
RESULTS AND CONCLUSION: Compared with simple high-temperature repairing for 10-20 minutes, the tissue structure was preserved completely after high-temperature repairing for 15 minutes with citric acid followed by antigen retrieval solution, and there was no significant difference in background staining; the blockage of normal serum with 10% concentration of second antibody under room temperature was regarded as the best conditions of antigen. Under this condition, we could guarantee a good sealing effect, and would not affect the binding of primary antibody and antigen sites. Higher concentration of primary antibody   (10 mg/L) could ensure a clear fluorescence signal, low background and acceptable cost. The paraffin sections were preformed with immunofluorescence assay, and observed with an ordinary fluorescence microscope. The FITC excitation light blue and green fluorescence signal marked dye groups were avoided. High-temperature repairing for 15 minutes with citric acid followed by antigen retrieval solution, close the normal serum with 10% concentration of second antibody under room temperature for       40 minutes, relatively high primary antibody concentration and observe under ordinary fluorescence microscope can improve the quality of immunofluorescence detection.