Plan apochromat 100

Fluorescence microscopy was performed with a Leica CTR6000 fluorescence microscope equipped with a Plan-Apochromat 100 × , 1.40 NA oil immersion objective lens. Images were taken using LAS AF 1.5.1 acquisition software (Leica). Cells were grown to early log phase (0.6 OD₆₀₀) in synthetic complete media and fixed by methanol/acetone before microscopy. Images were captured with a digital camera (DFC350FX; Leica) at room temperature.

CD14+ monocytes from three C9orf72-HRE patients and three FTD cases HRE negative were seeded onto Poly-L-lysine-coated glass coverslips by cytospin at 120 g for 5 min, fixed in 4 % paraformaldehyde and stained with 5’ TYE563-labelled [CCCGG]₃ probe (sense foci), or [GGGGCC]₃ probe (antisense foci) (Exiqon) as previously described [5 (link)]. Nuclei were stained with Hoechst and slides were subsequently imaged on a Leica TCS SP8 confocal microscope using a Leica Plan Apochromat 100× oil immersion objective lens. The resulting images were analyzed using an internally developed pipeline in CellProfiler [42 (link)]. Briefly, maximum projections of each image channel were made in Fiji [43 (link)] and used as input in the pipeline for at least 70 cells for each patient and probe. Threshold value was automatically set for each image and nuclei were detected. Speckles were then enhanced in the RNA foci channel, and this channel was subsequently masked based on the nuclei channel. Threshold was set per object basis using the Mixture of Gaussian algorithm. The RNA foci were then automatically counted and assigned to a nucleus, and the percentage of nuclei containing at least one focus was determined for each sample.