Alexa 594 conjugated anti mouse and anti rabbit antibodies

For all immunofluorescence procedures, we followed our earlier protocols (Peltonen et al., 2014a (link)). Cells grown on coverslips were fixed in 3.5% paraformaldehyde or 100% methanol, permeabilized with 0.5% NP-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, and 50 mM NaF), and blocked in 3% BSA. The following primary antibodies were used: POLR1B/RPA135 (4H6; Santa Cruz Biotechnology), POLR1A/RPA194 (C-1; Santa Cruz Biotechnology), NPM (FC-61991; Invitrogen), NCL (4E2; Abcam), and fibrillarin (ab5821; Abcam). Secondary antibodies used were Alexa 488 and Alexa 594-conjugated anti-mouse and anti-rabbit antibodies (Invitrogen). DNA was stained with Hoechst 33342. Images were captured using DM6000B wide-field fluorescence microscope (Leica). The microscope was equipped with a Hamamatsu Orca-Flash 4.0 V2 sCMOS camera and LAS X software by using 40×/1.25–0.75 HCX PL APO CS oil and 63×/1.40–0.60 HCX PL APO Lbd.bl. oil objectives. Quantitative image analysis of nucleolar protein expression was as described in Peltonen et al. (2014a) (link) and was conducted on at least 200 cells per sample on three to five fields.