PtK2 rat kangaroo kidney epithelial cells were grown in DMEM supplemented with 10% heatinactivated fetal calf serum at 37°C and 8% CO2. They were plated on Aclar plastics (EMS; Science Services, Munich, Germany) cut to fit perpendicularly in the baskets of the Leica EM AMW sample holder. The substrata were cleaned by HCl treatment for 2 hours and subsequent washing in distilled water and 70% ethanol. Before use, they were incubated in culture medium at 37°C overnight. Cells were grown 70% to 90% confluency and split 1:3 by use of trypsin/EDTA every third day.
1. The Leica EM AMW was prepared by filling the vials for fixation and washing steps with 10 ml of the appropriate liquids (see table), “START” was pressed to bring vial 1, containing 0.5 % glutaraldehyde in cacodylate buffer, in position for MW fixation.
2. Cells on Aclar were transferred from tissue culture dished to the baskets of the sample holder containing four divisions. To prevent drying of the samples, the baskets were immersed in phosphate buffered saline (PBS). Up to 4 Aclar substrata were placed perpendicularly into the divisions of a basket, and a second one was placed upside down to confine each sample individually. For this the divisions of both baskets were aligned to each other. Up to three pairs of baskets, providing altogether space for 12 samples, fit to the stem of the Leica EM AMW sample holder.
3. After mounting, the sample holder was transferred quickly into the Leica EM AMW. Immediately after immersion of the samples in the fixative, MW exposure was initiated by turning its lid for closure of the microwave chamber.
4. Subsequently, cells were washed in cacodylate buffer. Further processing, comprising osmification with 0.5% OsO4, dehydration in a series of ethanol, and infiltration with epoxy resin (Agar 100) was performed in a tissue processor Leica EM TP (For details see Reipert et al., 2008).
For these steps, the samples remained in their baskets, since they also fit to the stem holder of the Leica EM TP tissue processor. Polymerization was carried out in a standard oven, 60°C for 36h.