Whole-mount immunostaining that avoids cross-reaction between antibodies from different host species for simultaneous visualization of actin filaments and microtubules.

The plant cytoskeleton, composed of microtubules and actin filaments, is an essential structural element for plant growth and development; it optimizes cell size and shape along the differentiation trajectories. Thus, visualizing and observing the cytoskeleton's spatial organization within cells is crucial to better understanding plants' developmental strategies as sessile organisms. Here, we developed a whole-mount immunostaining method for double-labeling actin filaments and microtubules using Arabidopsis thaliana roots. To enable this, we examined the specificity of the secondary antibody toward the primary antibody raised in different host-species to propose two optimal methods to double-label actin filaments and microtubules, depending on the combinations of the host-species for primary antibodies: "simultaneous immunostaining", in which two sets of primary and secondary antibodies are applied simultaneously and "sequential immunostaining", where two rounds of antibody-antigen reactions are conducted sequentially. The sequential reaction aims to avoid cross-species immunoreaction, where the secondary antibody undesirably binds to the primary antibody from a different host species. Our findings can provide valuable information on how to select antibodies not only for the cytoskeletal elements but also for other proteins of interest.