Different methods of tissue preparation for X-ray microanalysis combined with scanning electron microscopy based on zebra fish, Brachydanio rerio muscle fibres

Four preparation methods for electron probe X-ray microanalysis (EPXMA) of muscle fibres were compared: (1) dissection, freezing in LN2 (liquid nitrogen), cutting in cryostat, freeze-drying and analysis; (2) dissection, immersing in Tissue-Tek, freezing in LN2, cutting in cryostat, the following steps as in method 1; (3) dissection, freezing in LN2, freeze-drying, separation of fibres into groups; (4) dissection, cutting into 2 mm thick slices by razor blade, freezing in LN2, following steps as in method 1. The contents of Na, Cl, and K as well as K/Na, Cl/K, ratios were taken as criteria of good preservation of muscle fibres. The best results were obtained by method 1. Good morphological preservation can be routinely observed in sections prepared by methods 3 and 4. However, the diffusible elements and K/Na, Cl/K ratios were not retained at relatively constant level among the individual samples. Fibres prepared by methods 1 and 3 showed, despite of freezing artefacts, high K/Na, and low Cl/K ratio. After method 1, high level of the elemental contents was retained, and it did not differ significantly among the samples, showing relatively low standard deviation values.