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Combined Autofluorescence Quenching

This combined method has been suggested to tackle autofluorescence from tissue elements including lipofuscin and formalin fixation. 

1. Dewax and rehydrate FFPE sections in xylene and decreasing concentrations of IMS.

2. Wash sections in dH2O for 5 min.

3. Heat-induced antigen retrieval (if required)

4. Wash sections in dH2O for 5 min, followed by TBS-T 0.1% for 2x 5 min.

5. Blocking with normal goat serum in TBS-Triton X 0.25% for 1 h.

6. Overnight primary antibody in TBS-Triton X 0.1% incubation at 4 C.

7. Wash sections in PBS (no detergents) for 2x 5 min.

8. Incubate with 0.3% Sudan Black 

9. Wash sections in PBS for 5 min.

10. Incubate with TrueVIEW for 5 min.

11. Wash sections in PBS for 5 min.

12. Incubate with fluorescent secondary antibodies in PBS for 1 h at room temp.

13. Wash sections in PBS for 2x 5 min.

14. Coverslip using VECTASHIELD Vibrance * and allow to set for 2 h.


*VECTASHIELD Vibrance included in TrueVIEW Kits, but may also be purchased separately