High-resolution microscopy

Images of immunofluorescence-labeled cell cultures were acquired using an AxioImager Z.2 microscope (Zeiss), equipped with wide-field optics, a ×20, 0.8 NA dry objective and a quadruple-band filter set for Hoechst, FITC, Cy3 and Cy5 fluorescent dyes. Wide-field acquisition was performed using the Colibri 7 LED light source and an AxioCam 702 mono camera with 5.86 μm per pixel. Z-stacks with 19 z-slices were acquired at 3-mm increments to capture the optimal focus plane. Images were obtained automatically with Zeiss ZEN 2.6 (blue edition) at non-saturating conditions (12-bit dynamic range).

IHC images from salivary gland and melanoma tissue were obtained using the automated slide scanner Zeiss Axio Scan.Z1 for bright-field microscopy. Bright-field acquisition was obtained using the VIS LED light source and a CCD Hitachi HV-F202CLS camera. PEN slides were scanned with a ×20, 0.8 NA dry objective yielding a resolution of 0.22 mm per pixel. Z-stacks with eight z-slices were acquired at 2-mm increments to capture the optimal focus plane. Color images were obtained automatically with Zeiss ZEN 2.6 (blue edition) at non-saturating conditions (12-bit dynamic range).

Cells were imaged on a Leica Dmi8 wide-field microscope equipped with a 0.8 NA, ×40 air objective and a Hamamatsu Flash 4.0 V3 camera using LAS X software. The segmentation of each cell was performed using Cell Profiler software⁸ using DAPI for nuclei segmentation. The mean intensity of the target protein and the cell cycle marker protein was measured in the nucleus. The cells were grouped into the G1 and G2 phases of the cell cycle by using the 0.2 and 0.8 quantile of ANLN or CCNB1 intensity levels in the nucleus, and cell-cycle-dependent expression of C7orf50 was validated by comparing differences in expression levels between G1 and G2 cells.