Table 1.
Staining protocol used in Experiments 1 and 2
| Process | Step | Solution | Duration (m:s) | Process | Step | Solution | Duration (m:s) | |
|---|---|---|---|---|---|---|---|---|
| Dewaxing | 1 | Xylene (wash 1) | 3:00 | Staining | 14 | Running tap water | 1:00 | |
| 2 | Xylene (wash 2) | 3:00 | 15 | Scott’s Tap Water | 2:00 | |||
| 3 | Xylene (wash 3) | 3:00 | 16 | Running tap water | 1:00 | |||
| 4 | Xylene (wash 4) | 3:00 | 17 | Aqueous Eosin Y (1%) |
0:15 − 6:00 (see SI Table 2) |
|||
| Rehydration | 5 | 100% ethanol (wash 1) | 3:00 | 18 | Running tap water | 1:00 | ||
| 6 | 100% ethanol (wash 2) | 3:00 | Dehydration | 19 | 100% ethanol (wash 1) | 0:30 | ||
| 7 | 100% ethanol (wash 3) | 3:00 | 20 | 100% ethanol (wash 2) | 1:00 | |||
| 8 | 100% ethanol (wash 4) | 3:00 | 21 |
100% ethanol (wash 3) |
5:00 | |||
| 9 | 75% ethanol | 3:00 | 22 | 100% ethanol (wash 4) | 5:00 | |||
| 10 | 50% ethanol | 3:00 | Clearing | 23 | Xylene (wash 1) | 3:00 | ||
| 11 | 25% ethanol | 1:00 | 24 | Xylene (wash 2) | 3:00 | |||
| 12 | Running tap water | 2:00 | 25 | Xylene (wash 3) | 3:00 | |||
| Staining | 13 | Mayer’s Haematoxylin | 0:15 − 6:00 (see SI Table 2) | Mounting | 26 | DPX mountant | - |
The staining protocol used in Experiment 1 and 2 is described, outlining process, solution and duration used. All steps in this protocol were undertaken at room temperature. Please note stain technique 1 (haematoxylin only) excluded steps 17–18, and stain technique 2 (eosin only) excluded steps 13–16. Stain technique 3 included all steps. Abbreviations: m:s, minutes : seconds; SI, Supplementary Information; DPX, Dibutylphthalate Polystyrene Xylene