Leica dm6000 fluorescent microscope

Three-dimensional images for distance measurements were taken on a Leica DM6000 fluorescent microscope (Leica Microsystems) with a DFC360FX-325642208 camera and a HCX PL APO CS 100.0×1.40 oil objective (voxel size 128:128:148 nm [x:y:z]). Distances were measured manually from the center of each BAC signal to the edge of the closest chromocenter (marked by satellite probe) using ImageJ software as previously described (Splinter et al. 2011 (link)). Association was called when distances between the center of the BAC signal and the edge of the closest chromocenter were <0.3 μm, the maximum distance at which visual associations (touching signals) have been observed in our measurements. For comparative measurements in lacO transgenic cells, Z-stacks were renamed in a randomized fashion to allow unbiased measurements, and only cells were included that showed two BAC spots and one overlapping lacO spot.
Imaging of EGFP-LacR fusions was performed on a Leica TCS SPE spectral confocal microscope (Leica Microsystems) using an ACS APO 63.0×1.30 oil objective with 3.0× zoom (voxel size 56.9:56.9:209.8 nm [x:y:z]).

Immunofluorescence staining was conducted as described previously (30 (link)). Primary antibodies used were mouse monoclonal anti-MYC-Tag 9B11 (1:750; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-HA Y-11 (1:100; Santa Cruz, Dallas, TX), mouse monoclonal anti-HA 16B12 (1:100; BioLegend, San Diego, CA), rabbit monoclonal anti-Na⁺/K⁺/ATPase (Alexa Fluor^® 488), EP1845Y (1:50; Abcam, Cambridge, United Kingdom), and rabbit monoclonal anti-Na⁺/K⁺/ATPase EP1845Y (1:250; Abcam).
A Zeiss LSM 510 confocal microscope with × 40 objective was used to perform confocal microscopy (Carl Zeiss AG, Oberkochen, Germany). Epifluorescent microscopy was performed using × 40 objective on a Leica DM6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The Duolink In Situ Fluorescence Protocol with Detection Reagents Red Kit (Sigma) was used as per the manufacturer's instructions. Cells were transfected for 48 hours before fixation, permeabilization, and addition of anti-MYC and anti-HA antibodies.