Meiotic Chromosome Immunofluorescence Imaging

Young anthers during meiotic stages were fixed in 4% (w/v) paraformaldehyde for 30 min at room temperature and stored in 1x Buffer A at 4°C. Immunofluorescence was performed as previously described (Pawlowski et al., 2003 (link); Cheng, 2013 (link)). The primary antibodies against ASY1, ZYP1, and γH2AX were prepared as described previously (Jing et al., 2019 (link)). Antibody against RAD51 was a gift from Wojtek Pawlowski’s Lab at Cornell University. Fluorochrome-coupled secondary antibodies (ABclonal) were used for fluorescence detection. All primary and secondary antibodies were diluted at 1:100. Images of meiocytes were observed and captured using a Ci-S-FL microscope (Nikon) equipped with a DS-Qi2 microscopy camera (Nikon, Tokyo, Japan). The images were captured by software NIS-Elements and colored by the ImageJ software.