Simultaneous triple staining for detecting cell-type specific spatio-temporal distribution of cell wall materials in monocot roots

Summary Anatomical and histochemical imaging of grass root systems relies on tissue sectioning and cell wall staining dyes because molecular reporter lines are limited for most organisms. Distinct staining dyes require variable incubation time and concentration across different tissues and organisms. As a result, staining with multiple dyes becomes time consuming or challenging. Here, we report a rapid method to perform simultaneous triple staining on a glass slide. The entire protocol requires ∼4 hours and a smaller volume of stain than traditional methods. We tested this method using the roots of two economically important crops, Triticum aestivum (wheat) and Zea mays (maize), as proof of concept. We have also demonstrated the presence of exodermis in wheat roots. Additionally, we identified the formation of polar lignin caps in maize exodermis using our simultaneous triple staining method. This method empowers a quantitative approach to cell biology by elucidating cell-type specific spatio-temporal distribution of cell wall materials in monocot root systems.