In-slide sample preparation allows for rapid triple staining and the detection of specific cell wall components in monocot roots

Abstract Anatomical and histochemical imaging of monocot 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 and clearing of samples 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 3 crops, Triticum aestivum (wheat), Zea mays (maize), and Cucurbita pepo (winter squash), and the stems of 4 crops, Cucurbita pepo (winter squash), Solanum lycopersicum (tomato), Tagetes patula (marigold), and Camelina sativa (false flax). We have demonstrated the presence of exodermis in wheat roots and a multiseriate exodermis in maize roots. Images produced with this method are compatible with ImageJ/Fiji based quantification methods. This sample preparation method ultimately simplifies the staining process while empowering a quantitative cell biology approach to study cell-type specific distribution of cell wall materials in grass root systems and beyond.