Table 1 Optimized staining protocol for ERα, CK, and CD45 expression in CTCs and leukocytes
Step | Reagent | Concentration | Interval | Manufacturer/Batch |
|---|---|---|---|---|
1. Cell fixation | Methanol | 1:1 | 5 min at RT | Merck KGaA, Germany, #I659409 |
2. Cell permeabilization | Dako Envision Target Retrieval solution™ (50x) (Tris/EDTA buffer solution, pH 9.0, and detergent) | 1:50 | 20 min at 37°C | Dako Denmark, A/S, #20000821 |
3. AB serum | Dako AB diluent™ (1% FBS in PBS, 0.1% detergent) | 1:1 | 20 min at RT | Dako Denmark, A/S, #00091216 |
4 1 . ERα labelling | Rabbit monoclonal AB specific to ERα | 1:50 | 60 min at 37°C | Thermo Scientific, United States, #9101S1210D |
5 1 . CD45 staining | Alexa Fluor 647 labeled mouse monoclonal AB specific to CD45 | 1:5 | 60 min at 37°C | AbD Serotec, UK, #B173123 |
6 2 . ERα staining | Alexa Fluor 488 labeled goat anti-rabbit secondary AB | 1:200 | 45 min at RT | Life Technologies, United States, #1423009 |
7 2 . Cytokeratin 8, 18, and 19 staining | Phycoerythrin labeled mouse monoclonal AB, specific to CK 8, 18, and 19 | 0.0006% | 45 min at RT | Janssen Diagnostics, United States, #E491A |
8. Nuclear counterstaining | Slowfade® Gold antifade with nuclear dye, DAPI | Life Technologies, United States, #1500156 |