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PD-L1 (SP142) from Spring Bioscience, Inc., with DISCOVERY HQ-HRP detection and ChromoMap DAB detection in non-small cell lung cancer (NSCLC)

Reagents Needed from Ventana

Name Catalog number Ordering code

DISCOVERY Anti-Rabbit HQ 760-4815 07017812001

DISCOVERY Anti-HQ HRP 760-4820 07017936001

DISCOVERY ChromoMap DAB Kit (RUO) 760-159 05266645001

DISCOVERY CC1 950-500 06414575001

Hematoxylin II 790-2208 05277965001

Bluing Reagent 760-2037 05266769001

Reagents Needed from Spring Bioscience, Inc.

Name Catalog number Dilution in protocol

PD-L1/CD274 (SP142) M4420 1: 30

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RUO DISCOVERY Universal Staining procedure for DISCOVERY ULTRA Research Instrument

VENTANA

Empowering | Innovation

www.roche.comwww.ventana.com

© 2014 Ventana Medical Systems, Inc.

VENTANA, VENTANA logo and DISCOVERY are trademarks of Roche. All other trademarks are the property of their respective owners. W5204-8 0814A

1. Deparaffinization [Selected]

2. Warmup Slide to [67 Deg C], and incubate for [8 Minutes] (Cycle 1)

3. Incubate for [8 Minutes] (Cycle2)

4. Incubate for [8 Minutes] (Cycle3)

5. Pretreatment [Selected]

6. Cell Conditioning [Selected]

7. CC1 Reservoir [Selected]

8. Warmup Slide to [100 Deg C], and incubate for 4 Minutes (Cell Conditioner #1)

9. CC1 8 Min [Selected]

10. CC1 16 Min [Selected]

11. CC1 24 Min [Selected]

12. CC1 32 Min [Selected]

13. CC1 40 Min [Selected]

14. CC1 48 Min [Selected]

15. CC1 56 Min [Selected]

16. CC1 64 Min [Selected]

17. CC1 72 Min [Selected]

18. CC1 80 Min [Selected]

19. CC1 88 Min [Selected]

20. CC1 92 Min [Selected]

21. Inhibitor [Selected]

22. Inhibitor CM [Selected]

23. Apply One Drop of Inhibitor CM, and Incubate for [12 Minutes]

24. Antibody [Selected]

25. Disable heat for 1st Antibody [Selected]

26. Apply One Drop of [PREP KIT 1] (Antibody), and incubate for [1 hour]

27. Linking Antibody [Selected]

28. 2nd Antibody [Selected]

29. Warmup Slide to [36 Deg C] from Very Low Temperatures (2nd Antibody)

30. Apply One Drop of [Anti-Rabbit HQ] (Detection #1), and incubate for [0 HR 16 Min]

31. Enzyme conjugate [Selected]

32. Apply One Drop of [Anti-HQ HRP] (Conjugate #1), and Incubate for [12 Minutes]

33. DAB [Selected]

34. Counterstain [Selected]

35. Use RB for Counterstain [Selected]

36. Apply One Drop of [HEMATOXYLIN II] (Counterstain), and incubate for [8 minutes]

37. Post Counterstain [Selected]

38. Use RB for Post Counterstain [Selected]

39. Apply One Drop of [BLUING REAGENT] (Post Counterstain), and incubate for [4 minutes]