Two interacting axis proteins contribute to meiotic stability in autotetraploid Arabidopsis arenosa

Slides were then mounted in 7 μL 1 μg/mL 4',6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector Laboratories). Immunocytology For immunostaining of A. arenosa pachytene cells, we followed the protocol described in (Higgins et al. 2014) with minor modifications. Briefly, anthers containing meiocytes of the desired meiotic stage were dissected from fresh buds and macerated on a No.1.5H coverslip (Marienfeld) in 10 µL digestion medium (0.4% cytohelicase (Sigma), 1.5% sucrose, 1% polyvinylpyrrolidone (Sigma) in sterile water) for 1 minute using a brass rod.

Coverslips were then incubated in a moist chamber at 37°C for 4 minutes before adding 10 µL of 2% Lipsol solution (SciLabware) followed by 20 µL 4% paraformaldehyde (pH 8). Once coverslips were dry they were blocked in 0.3% BSA in PBS and then incubated with primary antibody overnight at 4°C and secondary antibody for 2 hours at 37°C. Before and after each antibody incubation coverslips were washed in 1 X PBS solution plus 0.1% Triton X-100 (Sigma).

Coverslips were finally incubated in 10 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes before being mounted on a slide in 7 µL Vectashield (Vector Laboratories). The following primary antibodies were used at 1:500 dilutions: anti-ASY1 (rat and guinea-pig), anti-ZYP1 (rat and guinea-pig), anti-HEI10 (rabbit). The following secondary antibodies were used at 1:200 dilutions: anti-rat Alexa Fluor 488 (Thermofisher), anti-rat Alexa Fluor 555 (F(ab')2 fragment, Abcam), anti-guinea pig Alexa Fluor 488 (Thermofisher), anti-rabbit Alexa Fluor 647 (F(ab')2 fragment, Thermofisher).

Immunostained cells were imaged using 4-colour structured illumination microscopy (3D-SIM) on a Zeiss Elyra PS1 microscope.