Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment

Immunolocalizations in whole mounts of walled cells require an empirically derived, species-specific permeabilization strategy, Particularly when cell walls are recalcitrant to enzymatic digestion, as with many fungi, permeabilization can degrade structure and reduce or destroy antigenicity, To avoid these potential problems, hyphae were freeze-substituted, embedded in a methacrylate resin, thick-sectioned and de-embedded prior to immunolocalization. The cytoskeletal proteins actin and beta-tubulin were labelled via indirect immunofluorescence in hyphal tip cells of Magnaporthe grisea, and Trichoderma viride, Well-defined punctate actin plaques were observed concentrated at the subapical cell periphery in both species, especially near the hypha-substratum interface, In addition, a Spitzenkorper core and less intense cytoplasmic binding were observed. Immunoelectron microscopy confirmed localization of actin in the Spitzenkorper core and filasomes. beta-Tubulin was immunolocalized as both linear al-rays and punctate dots via epifluorescence microscopy, Analysis of 450-550-nm-thick serial sections contributed to improved resolution by the reduction of out-of-focus blur. Overall,tbe freeze substitution methacrylate de-embedment technique provided superior preservation, serial section analysis, and improved antibody penetration and resolution of these cytoskeletal proteins. The technique will be a valuable tool for use with a variety of reporter molecules in cells of fungi.