Yeast chromosome spreads were prepared as described (Xaver et al., 2013 (link)). Spread nuclei were stained by anti-myc 9E10 mouse antibody (1:50), followed by anti-mouse cy3-conjugated secondary antibody (Jackson ImmunoResearch, 1:400) for Spp1-myc or Bre2-myc, and with rabbit anti-HA antibody (Sigma, 1:100) followed by anti-rabbit FITC-conjugated antibody (Sigma, 1:500) for Mer2-HA. Immunostained, fixed chromosome spreads were analyzed on a ZEISS AXIO Imager M2, with a ZEISS Plan-Neofluar 100×, aperture: 1.3, and a 2× additional magnification by a Zeiss optovar. Specimens were mounted in Vectashield with 0.2 µg/ml DAPI, and well-spread nuclei were selected based on their DNA-morphology (DAPI). Images were taken at a constant exposure time of 2 s for DAPI (BFP channel), CY3 (CY3 channel), and FITC (FITC channel). Light source: Sola SM II (Lumencor); camera: CoolSNAP HQ2 (Visitron Systems GmbH); acquisition software: Visiview (Visitron Systems GmbH). From these records, the nuclear area, foci numbers and foci areas were determined using Fiji software. Signals were counted as overlaps if foci overlapped by >60% of their diameter.