Table 1.
Multiplex sequence order and control series
| Primary Secondary Antibodies | Negative Control | Cross Over Control 1 | Cross Over Control 2 | Cross Over Control 3 | Positive Control | |
|---|---|---|---|---|---|---|
| 1 | anti-aSMA | No (A) | Yes | Yes | Yes | Yes |
| anti-Mouse | Yes | No | Yes | Yes | Yes (H) | |
| 2 | anti-Vimentin | No (B) | No | Yes | Yes | Yes |
| anti-Mouse | Yes | Yes (E) | No | Yes | Yes (I) | |
| 3 | anti-K19 | No (C) | N/A | No | Yes | Yes |
| anti-Mouse | Yes | N/A | Yes (F) | No | Yes (J) | |
| 4 | anti-s100A4 | No (D) | N/A | N/A | No | Yes |
| anti-Rabbit | Yes | N/A | N/A | Yes (G) | Yes (K) | |
The final multiplex sequence is numbered and shows the application of primary and secondary antibodies for negative, crossover, and positive controls. Primary and secondary antibody pairs are highlighted. Each column corresponds to tissue section run through the entire multiplex labeling sequence (detection, scanning, elution and stripping performed between each antibody pair in sequence) unless otherwise specified (N/A). Yes: indicates reagent was applied. No: indicates antibody diluent substituted for primary antibodies. No: no secondary antibody reagent applied. Letters correspond to images shown in supplemental Figure 1.