Immunocytochemistry Protocol for TREx-BCBL-1-RTA Cells

TREx‐BCBL‐1‐RTA cells were seeded onto poly‐L‐lysine‐coated coverslips and 2 µg/ml doxycycline hyclate (Sigma‐Aldrich) was added 3 h later. Cells were fixed with 15 min with 4% paraformaldehyde and permeabilised with PBS + 1% Triton. All further incubation steps occurred at 37°C. Coverslips were blocked for 1 h with PBS and 1% BSA before 1 h incubation with the appropriate primary antibody and followed by 1 h with Alexa‐Fluor conjugated secondary antibody 488 (Invitrogen 1/500). Coverslips were mounted using Vectashield Hardset Mounting Medium with DAPI (Vector laboratories). Images were obtained using a Zeiss LSM880 Inverted Confocal Microscope and processed using ZEN 2009 imaging software (Carl Zeiss) as previously described (Baquero‐Perez & Whitehouse, 2015 (link)).