Immunocytochemistry and CLSM Analysis

Immunocytochemistry and CLSM were performed as previously described [74 (link),75 (link)]. Cells were cultured on 12 mm round coverslips coated with poly-D-Lysine/Laminin (BD Bioscience, Franklin Lakes, NJ). Cells were fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were incubated in a blocking buffer containing 1% bovine serum albumin (for MZF1 and SCAND2) or, alternatively, 3% normal goat serum (for SCAND1) in PBS for 30 min, incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at RT for 1 h in the blocking buffer. Cells were washed thrice with PBS for 5 min between the steps. Cells were mounted within ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence images were acquired using Axio Vision CLSM (Zeiss, Oberkochen, Germany) with an AxioCam MR3 (Zeiss) camera for SCAND1, and alternatively, FSX100 inverted microscope (Olympus, Tokyo, Japan) for MZF1 and SCAND2. We used antibodies against MZF1 (C10502, Rb pAb, Assay Biotechnology, Fremont, CA), SCAND1 (ab64828, Rb pAb, Abcam), SCAND2 (5F1, H00054581-M02, Ms mAb, Thermo Fisher Scientific), and anti-rabbit IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific).