SCREENING METHODS: Transmission electron microscopy(1)

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electron microscopy(1)

The specimens are then fixed with modified Karnovsky’s fixative, consisting of 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.05 M sodium cacodylate buffer solution (pH 7.2) at 4 °C for overnight. Subsequently, the fixed specimens are washed with the buffer solution three times for 10 min each. The specimens were then will be post-fixed in the osmium tetroxide solution (1% w/v) at 4 °C for 2 h followed by washing with millipore water twice each.

The post fixed specimens will be enbloc stained with 0.5% (w/v) uranyl acetate at 4 °C overnight and then will be dehydrated once in a graded ethanol series of 30, 50, 70, 80, and 95% and three times in 100% ethanol for 10 min each. The specimens will be further treated with propylene oxide twice for 30 min each as a transitional fluid and then will be embedded in Spurr’s resin. Ultra-thin sections (approximately 50 nm in thickness) will be cut with a diamond/ glass knife using an ultra-microtome. The sections will be mounted on copper grids and will be stained with 2% uranyl acetate and Reynolds’ lead citrate (Reynolds, 1963) for 7 min each.

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