Immunolocalization on whole anther chromosome spreads for male meiosis

Immunolocalization of cells undergoing meiosis has proven to be one of the most important tools to decipher chromatin-associated protein dynamics and causal relationships. Here, we describe a protocol established for maize which is easily adaptable to other plants, for example, with minor modifications to Arabidopsis as stated here. In contrast to many other protocols, the following protocol is based on fixation by a 3:1 mixture of ethanol and acetic acid. Spreading of cells is followed by freeze-shattering, protein antigenicity retrieval by a hot citrate buffer bath, antibody incubations and washes, and DNA staining.