A simple and robust protocol for immunostaining Arabidopsis pollen nuclei.

Pollen represents the male sexual lineage in flowering plants. At maturity, pollen grains are composed of a companion vegetative cell with embedded sperm. During pollen development, these two cell types acquire vastly differing cell fates. Underlying this differential fate acquisition is dramatic reconfiguration of pollen chromatin that is highly evident at a cytological level. The precise link between histone mark deposition and fate acquisition remains largely unexplored, which in part has been hindered by the difficulty in working with pollen in model plant species like Arabidopsis. Here, we describe a simple and robust protocol to isolate Arabidopsis pollen nuclei and immunostain for histone marks. Plant growth aside, the protocol can be performed over 2 days with few Arabidopsis plants, thus allowing multiple genotypes to be analysed in parallel. We also describe a method to de-mask epitopes through antigen retrieval, which vastly improves the signal for antibodies that target heterochromatic histone marks.