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In filamentous fungi a well-preserved hyphal cytoskeleton was obtained for indirect immunofluorescence (IIF) microscopy by quick-freezing in Freon 22 cooled with liquid nitrogen, followed by fixation with glutaraldehyde in ethanol or formaldehyde in methanol at −75°C. Several factors previously claimed to alter the structure of the cytoskeleton during the fixation process were totally or partially avoided. No microtubule stabilizing buffers were used, detergent treatment of hyphae was unnecessary when the fixative was formaldehyde in methanol, and the visualization of the microtubule cytoskeleton was possible without enzymatic treatment of hyphal walls. The procedure confirmed the picture of the structure of the microtubule cytoskeleton in the hyphae earlier achieved by more conventional fixation methods, and made possible the resolution of some new details. The visualization of actin was also improved.