Duolink reagents kit

Cells seeded on chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System), were fixed in 4 % paraformaldehyde for 10 min at 37 °C. Slides were then blocked in 3 % bovine serum albumin (Sigma) in a humidity chamber for 1 h at 37 °C and incubated overnight at 4 °C with mouse and rabbit antibodies: custom monoclonal anti-BORIS and rabbit anti-CTCF (D31H2) (Cell Signaling) in blocking solution. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS probes (Olink Bioscience). Ligation and detection were performed using the Duolink reagents kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence was detected using a Zeiss Plan Apochromat microscope with a ×63/oil objective. Images were acquired with an Axiocam MRm camera and Imaris software (Bitplane, Co.). The original microscopic images were deposited to Zenodo [83 ] (10.5281/zenodo.21405).

Frozen human testis slides were fixed in 4% paraformaldehyde for 10 min at 37 °C. Slides were then permeabilized for 2 h with 0.2% Triton X-100 in PBS then blocked in 3% bovine serum albumin (Sigma) in a humid chamber for 1 h at 37 °C and incubated overnight at 4 °C with mouse and rabbit antibodies: custom mouse monoclonal anti-BORIS and rabbit anti-TAF7L (Santa-Cruz, S-24) in blocking solution. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS probes. Ligation and detection were performed using the Duolink reagents kit (Olink Bioscience, Sigma) according to the manufacturer’s protocol. Fluorescence was detected using a Zeiss Plan Apochromat microscope with a 63x/oil objective. Images were acquired with an Axiocam MRm camera and the Zeiss LSM Image Browser. Presence or absence of foci in specific testis cell types was not assessed.