1.4 plan apochromat objective

Coverslips coated with Oregon Green 488-conjugated gelatin (Life technologies, ThermoFisher) were prepared as described in Thuault et al. [30 (link)]. After seeding for 4 h on gelatin-coated coverslips rehydrated in complete growth medium for 1 h before use, cells were fixed with a solution of 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA. When EB1 was labeled, cells were fixed with methanol before a 2nd fixation step with 4% formaldehyde in PBS and blocking with 1% BSA. Antibodies directed against the target proteins and secondary antibodies labeled with DyLight 405 or AlexaFluor 594 (Jackson ImmunoResearch, West Grove, PA, US) were then used for immunolabeling. A Zeiss structured light ApoTome microscope equipped with a 63× 1.4 plan ApoChromat objective was used for image acquisition (Zeiss, Jena, Germany). Ten random fields per coverslips, using 2 coverslips per condition per experiment, were imaged to assess the percentage of cells degrading. A home-made Fiji macro was used to analyze matrix degradation. The mean degraded area and the number of degradation foci per cell were quantified for 25 cells per condition per experiment.

Cells were plated on imaging slides (Ibidi), fixed with 4% paraformaldehyde, washed twice with PBS, and permeabilized with 70% ethanol overnight at 4 °C. After two washes with PBS and pre-hybridization solution (10% deionized formamide (Merck Millipore), 2x SSC), slides were incubated with 50 μl hybridization solution containing 2x SSC, 10% formamide, 50 μg competitor E. coli tRNA (Roche Diagnostics), 10% dextran sulfate (VWR), 2 mg/ml BSA (UltraPure; Life Technologies), 10 mM vanadyl-ribonucleoside complex (NEB) and 1 ng/μl smFISH probes for 6 h at 37 °C. Afterwards, slides were washed twice with pre-hybridization solution at 37 °C, then twice with PBS with subsequent mounting with ProLong® Gold Antifade Reagent with DAPI. Slides were imaged after 12 h when the mounting medium was fully cured on an Axio Observer.Z1 inverted epifluorescence microscope equipped with a × 63/1.4 Plan-APOCHROMAT objective (Zeiss).
Probe Designer software by Biosearch Technologies was used to design probes for hNEAT1 5′ segment and mNEAT1 middle segment, both conjugated to Quasar®670 fluorescent dye. Sequences are listed in Additional file 5: Table S4. Probes for hNEAT1 middle segment, mNEAT1 5′ segment, and MALAT1 (all conjugated to Quasar®570) were pre-designed by Biosearch Technologies.