For analysis of CENH3 loading in homozygous mutants and WT, 2-week-old seedlings were used. Slides were prepared using a cytospin and used for immunostaining as it was described by Ahmadli et al. (2022b) . To determine the colocalization of βKNL2-EYFP protein with CENH3, immunostaining of nuclei/chromosomes with anti-CENH3 and anti-GFP antibodies and microscopic analysis of fluorescent signals were performed as previously described (Lermontova et al. 2013 (link)).
For time-lapse microscopy, seedlings of transformants were grown in cover slip chambers (Nalge Nunc International) for 7–10 days and analyzed with an LSM 510 META confocal laser scanning microscope (Carl Zeiss GmbH).
To investigate the interphase nucleus and centromeric chromatin ultrastructures at an optical lateral resolution of ∼100 nm (super-resolution achieved with a 405-nm laser excitation), we applied spatial structural illumination microscopy (3D-SIM) using a 63/1.40 objective of an Elyra PS.1 super-resolution microscope system (Carl Zeiss GmbH; Weisshart et al. 2016 ; Kubalova et al. 2021 (link)) DAPI (whole chromatin) and rhodamine (CENH3 signals) were excited by 405 and 561 nm lasers, respectively.
For time-lapse microscopy, seedlings of transformants were grown in cover slip chambers (Nalge Nunc International) for 7–10 days and analyzed with an LSM 510 META confocal laser scanning microscope (Carl Zeiss GmbH).
To investigate the interphase nucleus and centromeric chromatin ultrastructures at an optical lateral resolution of ∼100 nm (super-resolution achieved with a 405-nm laser excitation), we applied spatial structural illumination microscopy (3D-SIM) using a 63/1.40 objective of an Elyra PS.1 super-resolution microscope system (Carl Zeiss GmbH; Weisshart et al. 2016 ; Kubalova et al. 2021 (link)) DAPI (whole chromatin) and rhodamine (CENH3 signals) were excited by 405 and 561 nm lasers, respectively.