*2.9. Confocal Microscopy*

HFAs or U251 cells on coverslips were fixed for 15 min at room temperature with 4% electron microscopy grade paraformaldehyde (Electron Microscope Sciences; Hatfield, PA) in PBS. Samples were washed three times in PBS and incubated in blocking buffer (0.2% Triton X-100 (VWR Internationals; Radnor, PA, USA) and 3% bovine serum albumin (BSA; Sigma Aldrich; St. Louis, MO, USA) in PBS) at room temperature for 1.5 h. Incubations with primary antibodies diluted (1:1000) in blocking buffer (3% BSA and PBS) were carried out at room temperature for 2 h, followed by three washes in PBS containing 0.1% BSA. Samples were then incubated with secondary antibodies in Blocking buffer for 1 h at room temperature, followed by three washes in PBS containing 0.1% BSA. The secondary antibodies were Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 546 donkey anti-rabbit, and Alexa Fluor 647 chicken anti-goat (Invitrogen; Carlsbad, CA, USA). Prior to mounting, samples were incubated with 5 μg/mL DAPI (4',6-diamidino-2-phenylindole) for 5 min at room temperature before washing in PBS containing 0.1% BSA. Coverslips were mounted onto microscope slides using ProLong Gold antifade reagen<sup>t</sup> with DAPI (Invitrogen; Carlsbad, CA, USA). Samples were examined using an Olympus 1 × 81 spinning disk confocal microscope equipped with a 60×/1.42 oil PlanApo N objective. Confocal images were acquired and processed using Volocity 6.2.1 software.
