Table 2. Example of controls for a 5-marker multiplex immunofluorescence.
Eleven serial sections of a control block are stained with the experimental samples and serve as controls.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
|---|---|---|---|---|---|---|---|---|---|---|
| xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx | xxxxx |
| Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | Marker 1 | |
| Goat αMouse | GoatαMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse | Goat αMouse |
| Marker 2 | Marker 2 | Marker 2 | Marker 2 | Marker 2 | Marker 2 | Marker 2 | Marker 2 | Marker 2 | ||
| Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | |
| Marker 3 | Marker 3 | Marker 3 | Marker 3 | Marker 3 | Marker 3 | Marker 3 | Marker 3 | |||
| Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | Donkey αMouse | ||
| Marker 4 | Marker 4 | Marker 4 | Marker 4 | Marker 4 | Marker 4 | Marker 4 | ||||
| Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | Donkey αRabbit | |||
| Marker 5 | Marker 5 | Marker 5 | Marker 5 | Marker 5 | Marker 5 | |||||
| αMouse IgG3biot | αMouse IgG3biot | αMouse IgG3biot | αMouse IgG3biot | αMouse IgG3biot | αMouse IgG3biot | αMouse IgG3biot | ||||
| 5 markers Qred | No primary control | No primary control | No primary control | No primary control | No primary control | 1 marker no Qred | 2 markers no Qred | 3 markers no Qred | 4 markers no Qred | 5 markers no Qred |
| stability | ||||||||||
| Controls for crossbinding | Controls for the loss of fluorescence during the full staining process | |||||||||
For stability test, on day zero, 20 (x20 objective) to 40 fields (x40 objective) of slide 1 are imaged using Nuance. At regular intervals (at least once a day for the time of the capturing), 4 fields are re-captured and the intensity of fluorescence for each marker is compared with day zero. The fields chosen are changed every day to avoid potential confounding from photobleaching (which does not happen in our current experimental conditions). Comparisons of slides 1 to 6 are used for cross binding control and 7 to 11 for quenching during the staining protocol.