Immunofluorescence localisation of microtubules in plant root tips embedded in butyl-methyl methacrylate

Most immunofluorescence studies of microtubules in plant cells have used enzymatically isolated cells whose position in the organ and stage of development was unknown. Moreover, attempts to label microtubules in cells in intact tissue have suffered from poor resolution and inferior tissue preservation. To overcome these difficulties, I have used a removable embedding resin to localise microtubules in situ. Tissues were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde and embedded in butyl-methyl methacrylate. Semi-thin sections were extracted with acetone to remove the resin and labelled with anti-tubulin followed FITC-labelled second antibody. Brightly stained microtubule arrays were clearly visualized. This reversible embedding medium should provide a powerful tool to study developmental changes in microtubule arrays in growing and differentiating plant tissues.