Melanoma Cell TEM Preparation

The samples for transmission electron microscopy (TEM) were prepared by incubating the confluent culture of melanoma cancer cells with the concentration of about 5 x 10⁶ in the presence of our tag structures with the cell to tag ratio of 1:5 in an 8-well Lab-Tek chambered cover-glass dish. After 18 h incubation period under standard conditions (37°C, 5% CO2, humidified), the samples were prepared for TEM sectioning. The preparation procedure [17 (link)] started with fixing the samples with fixative solution at room temperature for about 40 minutes. The solution was made of 0.1 M Sodium Cacodylate (pH 7.4) with the same percentages of 2% glutaraldehyde and 4% p-formaldehyde. The samples were then fixed again in 1% osmium tetroxide solution. After rinsing the samples with distilled water, the samples were stained with uranyl acetate. The samples then were dehydrated in ethanol solution. Finally the samples filtered and embedded in Epon for manual sectioning with a diamond knife. The sections were then imaged with JEOL JEM1400 images using a Gatan Orius model 832.