F ab 2 goat anti mouse igg alexa 594

Transduced K562 cells (1 × 10⁶) were fixed with an equal volume of 4% (w/v) formaldehyde for 20 min at room temperature (RT). Cells were centrifuged at 2000 rpm for 3 min and resuspended in PBS twice. Cells (5.0 × 10⁵) were allowed to settle onto coverslips coated with poly-d-lysine, before drying and permeabilisation with Triton X-100 0.5% (v/v) in PBS for 10 min at RT. Cells were rinsed three times in PBS and blocked in 3% (w/v) BSA/PBS for 40 min at RT. Cells were rinsed three times in PBS and incubated with mouse anti-HA antibody (1:500, HA.11, Covance) for 90 min at RT. Cells were rinsed three times in PBS and incubated with F(Ab’)2-goat anti-mouse IgG-Alexa 594 (1:500, #A11020, ThermoFisher Scientific) and DAPI (1:10,000, #D1306, Life Technologies) for 40 min at RT. Cells were rinsed three times in PBS and mounted using Prolong Gold Antifade (Life Technologies). Slides were imaged at 60 × using the DeltaVision Personal (Applied Precision) and the DAPI, FITC and A594 filters. Images were analysed after deconvolution using Volocity software.