Table 2.

Optimized labeling protocol.

1. Prepare cytospin slide of enriched cell sample

2. Wash 1 × 5 min in PBST (PBS + 0.05% Tween 20)

3. Add permeabilization/blocking solution (PBS + 2% normal goat serum + 2% normal donkey serum + 1% BSA + 0.1% gelatin + 0.1% Triton X-100 + 0.05% Tween 20) for 30 min at room temperature

4. Add primary anti-CD45 (rabbit, polyclonal, 1:100) and primary anti-phospho-histone-H2A.X (Ser139; mouse, clone JBW301, 1:100) in antibody diluent, background reducing (Dako, Carpinteria, CA, USA) for 1 h at RT

5. Wash 3 × 5 min in PBST

6. Add secondary Alexa Fluor 647 donkey anti-rabbit IgG (H + L) and secondary Alexa Fluor 594 goat anti-mouse IgG (H + L) in PBST for 1 h at RT

7. Wash 2 × 5 min in PBST

8. Add custom-conjugated cytokeratin 8, 18, 19-Alexa Fluor 488 (clone CK3-6H5, 1:100) in 1% BSA/PBS for 1 h at RT

9. Wash 2 × 5 min in PBST

10. Mount with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA)