Dmi8 inverted widefield microscope

For immunofluorescence HeLa cells were seeded on glass cover slips and grown in 12-well plates. Cells were transfected with FLAG-FHL2 and/or HA-p57-wt encoding plasmids using PEI (polyethylenimine) as transfection reagent^{31 (link)}. After 30 hours cells were fixed using 4% paraformaldehyde and permeabilised with 0.1% Triton X-100 (#3051, Roth). After blocking with 0.5% gelatin (G1890, Sigma-Aldrich), samples were incubated with the following primary antibodies: rabbit polyclonal p57 ((C20) sc-1040, Santa Cruz) and monoclonal mouse anti-FLAG (M2 #F3165, Sigma-Aldrich). After washing, cells were incubated with fluorescence labeled antibodies 488 and 555 (Alexa Fluor, Invitrogen). Cells were mounted in a DAPI containing mounting medium (ab104139, Abcam). Analysis was performed on a Leica DMi8 inverted widefield microscope.
For fluorescence microscopy HeLa cells were grown in chambered coverslips (#80826, IBIDI, Planegg, Bavaria, Germany). Cells were transfected with Cherry-FHL2 and/or YFP-p57-wt or p57-Nt/p57-Nt-NLS1 encoding plasmids using PEI or Polyfect as transfection reagent. YFP (yellow fluorescent protein) was excited at 510 nm and mCherry at 550 nm. Analysis was performed on a Leica DMi8 inverted widefield microscope.

Long-term cultures in 48-well, laminin-coated plates were used for immunocytochemistry (ICC) analysis. After aspirating culture medium, wells were rinsed with PBS, and cells were fixed in 4% PFA for 20 min at room temperature. Cells were then incubated in 2% normal goat serum (NGS; MilliporeSigma, G9023) with 0.1% Triton X-100 (MilliporeSigma, X100) in PBS to permeabilize and block for 30 min at room temperature. Primary antibodies (see Table 2) were diluted in 1% NGS in PBS and cells were stained overnight at 4 °C. Wells were washed 3 times for 10 min per wash with PBS. Secondary antibodies (see Table 3) were diluted in 1% NGS in PBS and filtered through a 0.22 µm PVDF syringe filter (MilliporeSigma, SLGV033RS). To prevent fluorescence photobleaching of conjugated secondary antibodies, plates were wrapped in foil, while cells were incubated for 1 h at room temperature. Secondary antibody solutions were removed, and cells were washed 3 times for 10 min per wash with PBS. Cells were then stained in 1:1000 Hoechst 33258 (Thermo Fisher Scientific, H3569) in PBS for 10 min, washed once with PBS, then imaged on a DMi8 inverted widefield microscope (Leica).