3D multiple immunoimaging using whole male organs in rice

Abstract

Spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a prominent part of the male reproductive organ in plants, contains several somatic cell layers named the anther wall and, within it, the germ cells. Here, we successfully developed a simple 3D organ-immunoimaging technique for rice anthers, which distinguishes each individual cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing. The 3D immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes, as exemplified by MEL1, a germ cell-specific ARGONAUTE in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our 3D multiple immunostaining method with single-cell and intracellular resolution will contribute to a comprehensive organ-level elucidation of molecular mechanisms and cellular connectivity.

Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway

“…2a and Supplementary Data 2 ). Recently, we developed a 3D-anther immunostaining system at subcellular and single-cell resolution 31 . To investigate the localization of these AGO1b/d proteins in anthers, we performed the 3D multiple immunostaining against AGO1b and AGO1d using whole-mount anthers at early meiosis.…”

“…The slide was incubated in a dark chamber for 2 h at room temperature, and then incubated overnight at 4 °C. The samples were washed three times with PME buffer for 5 min, incubated for 15 min at 25 °C in 1 µg/mL DAPI (Sigma, MBD0015), and washed again with PME buffer as above 31 . Samples were mounted in ProLong Gold antifade reagent (Invitrogen, P10144).…”

“…The samples were incubated for 15 min at 25 °C in 1 µg/mL DAPI diluted 1/1000 with PME (Sigma, MBD0015), followed by washing three times with PME buffer. Samples were mounted in ProLong Gold antifade reagent (Invitrogen, P10144) and images were captured using an LSM 880 microscope (Carl Zeiss) 31 .…”