Immunofluorescence Staining Protocol for U87 Cells

U87 cells (grown for 48 h on glass slides or following cytospin at 400 rpm for 3 min) were fixed for 30 min in 4% paraformaldehyde in PBS. For actin staining, cells were permeabilized and blocked with 0.1% saponin (Sigma) and 0.1% BSA (MD Millipore-Merck, Burlington, MA, USA) in PBS–Tween (PBS-T) and incubated for 1 h with Flash Phalloidin Green 488 (1:100; BioLegend, San Diego, CA, USA) in the dark [16 (link)]. For LRP1B expression analysis, cells were permeabilized with 0.5% Triton X-100 in PBS, blocked with 3% BSA and 0.1% Triton X in PBS, and incubated with LRP1B antibody (1:500 in blocking buffer; Sigma SAB4200326) overnight at 4 °C. After washing with PBS, slides were further incubated with secondary anti-rabbit antibody (Alexa 594, Invitrogen A-11037) for 1h at room temperature. All slides were incubated with DAPI (1:1000) for 5 min, washed, and mounted with Vectashield antifade mounting medium (Vector Laboratories). Fluorescence was analyzed using LEICA DM 2000 or with Zeiss Axio Imager Z1 Apotome microscopes with a coupled camera. Nuclear morphometric analysis (NMA) was carried out in images from DAPI-stained nuclei (with at least 300 dpi) using ImageJ analysis software with NII Plugin [82 (link)].