- PTE cells are grown on coverslips previously treated with Collagen S (see preparation of matrix substrate)
- At approx. 80% cell confluent, remove culture medium by suction. Wash cells 3x with PBS and fix them with acetone or methanol at -20oC for 5-10 min. Remove fixative from the chambers by suction and wash cells 3x with PBS
- Block unspecific binding by adding undiluted fetal calf serum. Incubate for 15 min 37oC in a closed moist chamber. 1% BSA (w/v) in PBS can also be used as blocking reagent.
- Wash several times with PBS to remove blocking reagent. Remove excess PBS and incubate preparation for 60 min at 37oC with 20-50 ul monoclonal anti -pan cytokeratin (clone C-11, mouse IgG1 isotype, Sigma, Cat. no. C2931) in a humidified chamber.
- Wash shortly 6x with PBS, remove excess PBS and incubate preparation with 20-50 ul of FITC-conjugated anti-mouse secondary antibody (concentration 20-50 ug/ml) for 60 min at 37oC.
- Wash shortly 6x with PBS and 6 times in redistilled water and embed the moist sample in mounting medium (eg. 180 ul of 1:2 glycerol:PBS pH 8.5 + 20 ul 1% 14-phenyldiamine or commercial fluorescent mounting medium from Dako).
- To embed coverslip put one droup of mounting medium on a slide and place coverslip (the side with the cells) into the drop. Seal with a high-tech and expensive reagent called nail polish.
- Viewed under standard fluorescence microscopy.
- Used mouse IgG1 as a control
- PTEC should be positive for cytokeratin, negative for vimentin and the distal tubular marker Tamm-Horsfall protein.
