Mounting Technique Allows Observation of Immuno-Labeled Cells on Plastic Coverslips

Some cells grow very poorly on glass, if at all, unless its surface has been coated with a polymer (e.g., polylysine, collagen, agarose etc.) to permit cell adhesion (2). Unfortunately, these polymers can also nonspecifically bind primary or secondary antibodies, which greatly increases the background. To address this problem, we have used Thermanox plastic coverslips (Nalge Nunc International, Rochester, NY, USA) to grow 16HBE lung cancer cells. These coverslips are characterized by nonidentical faces, one of which is modified to increase cell adherence. This material is not recommended for immunofluorescence assays, as specified by the manufacturer, partly because of the strong autofluorescence of the plastic and partly because of its low light transmission capacity. After fixation and labeling with fluorochromes, the coverslips were carefully loaded onto a drop of an aqueous medium on the surface of a glass slide using a technique widely used for glass coverslips and referred to here as the standard protocol (Figure 1A). Alternatively, we have developed a technique in which the plastic coverslips are first fixed on the glass slide, loaded with a drop of aqueous medium and then covered with a glass coverslip (Figure 1B). This new technique considerably reduces the effects described above and allows the detection on plastic coverslips of specific antigens and DNA using any fluorochrome. Its main advantages are that it is simple, cheap and easy to carry out. We have detected the Ki-67 antigen, a human nuclear protein whose expression is strictly associated with cell proliferation (3). This marker is widely used in routine pathology to measure the growth fraction of cells in tumors (1). The total number of cells was assessed by DNA staining, using either 4′,6-diamidino-2-phenylindole (DAPI) or chromomycin A3 (Sigma Chemical, St. Louis, MO, USA). 16HBE cells were grown on Thermanox coverslips in Dulbecco’s modified Eagle medium containing 10% fetal calf serum, 100 U/mL penicillin and 50 μg/mL streptomycin (Eurobio, Les Ulis, France) at 37°C in an atmosphere enriched with 5% CO2. Cells at a density of 5–10 × 104 per coverslip (22 mm in diameter) were cultured for 72 h. After several washings in phosphate-buffered saline (PBS), pH 7.2, the cells were fixed for 4 min in 3% (wt/vol) paraformaldehyde, 1% (vol/vol) Triton X-100 in PBS. The coverslips were then incubated for 30 min in PBS containing 3% (wt/vol) bovine serum albumin (BSA) (Sigma Chemical) and placed for 30 min with Mib1 monoclonal antibodies (Immunotech, Marseille, France) (6) diluted 1:20 in PBS containing 1% (wt/vol) BSA. The cells were washed in PBS (3 times, 5 min), and subsequently, biotinylated goat anti-mouse antibodies (Jackson, West Grove, PA, USA) (1:50 in PBS containing 1% [wt/vol] BSA) were applied for 30 min. After washing, the secondary antibody was detected