Ultrastructural immunolabelling of Penicillium marneffei

Department of Histopathology, Queen Mary Hospital, ^#Electron Microscopy Unit and ⁺Department of Microbiology, University of Hong Kong, Hong Kong.

For the immunoelectron microscopy, difficulties are commonly encountered on the preservation of the ultrastructure and the antigenicity at the same time. The factors such as reagents, concentration, duration, and temperature for each of the fixation, dehydration, infiltration and embedding, and antigen detection, have to be varied in order to optimise the results. In this study, the subcellular localization of an abundant antigenic candidate cell wall protein MpIp of pathogenic dimorphic fungus Penicillium marneffei was determined with a specific antibody raised against this recombinant protein. Different fixatives were assessed while the duration of the fixation, the dehydration with series of ethanol, the embedding with LR White resin and the signalling with gold-conjugated secondary antibody were applied constantly. It is concluded that the best fixation method for the ultrastructural immunolocalization of Penicillium marneffei is by suspending in 4% paraformaldehyde plus 2% glutaraldehyde in 0.1M phosphate buffer pH7.4 for 30 minutes at room temperature.

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