Micro cover glasses

Hemocytes were allowed to adhere (90 min, RT) to micro cover glasses (Matsunami, Osaka, Japan) in 6-well plates (BM Equipment, Tokyo, Japan). After removing the ASW, hemocytes were blocked (30 min, RT) with Ca²⁺/Mg²⁺-free ASW (CMFASW) containing 1% BSA; the composition of CMFASW was 480 mM NaCl, 9.4 mM KCl, 32 mM Na₂SO₄, 3.2 mM NaHCO₃, 10 mM EPPS, pH 8.0. The primary antibody (mAb11B16B10) and secondary antibody (Alexa Fluor 488 rabbit anti-mouse IgG; Molecular Probes, Thermo Fisher Scientific, Waltham, MA) were diluted to 1 and 0.4 μg/mL, respectively, in 2 mL of CMFASW containing 1% BSA, and added sequentially to each well and incubated (30 min, RT); the hemocytes were washed five times with CMFASW containing 1% BSA between incubations, and three times with ASW after incubation with the secondary antibody, and then examined under a fluorescence microscope (AXIO Imager M1; Zeiss, Oberkochen, Germany).

Micro cover glasses (MATSUNAMI, 18 × 18 mm, 0.12–0.17 mm) were placed in 35 mm dishes and coated with 1 ml of collagen matrices. Cell mixtures were cultured on these collagen matrices for 24 hr at 37 °C until a monolayer was formed. Dox (2 μg/ml) was added for 18 hr [mixed culture of normal MDCK plus v-Src cells or v-Src plus YAP (5SA) cells] or 21 hr [mixed cultures of normal MDCK plus YAP (5SA) cells]. Cells were washed three times in cold PBS and fixed in methanol for 2.5 min at −20 °C. Fixed cells were blocked for 1 hr in 1% BSA/PBS, and incubated with primary Abs for 16 hr at 4 °C, and then incubated with Alexa-488-conjugated secondary Abs for 1hr at room temperature. Immunostained cells were incubated with Hoechst dye in 1% BSA/PBS for 15 min and mounted with Mowiol on cover glasses (MATSUNAMI, 24 × 60 mm, 0.12–0.17 mm). Immunofluorescent images were captured and analyzed using a LSM710 Zeiss confocal microscope.

Micro‐cover glasses (Matsunami, 18 × 18 mm, 0.12–0.17 mm) were placed in 35‐mm dishes and coated with 1 ml of a collagen matrix. Cell mixtures (typically normal MDCK cells plus YAP (5SA) cells mixed at a ratio of 1:50) were cultured on these collagen matrices for 24 hr at 37°C until a monolayer was formed. Dox (2 μg/ml) was added for 22 hr to induce cDNA expression. Cells were washed three times in PBS, fixed with 4% paraformaldehyde/PBS for 15 min at room temperature and incubated with 0.5% Triton X‐100/PBS for 15 min at room temperature. Fixed cells were blocked for 1 hr in 1% BSA/PBS, followed by incubation with primary Abs for 16 hr at 4°C, and then with Alexa‐488‐conjugated secondary Abs for 1hr at room temperature. Immunostained cells were incubated with Hoechst dye in 1% BSA/PBS for 15 min and mounted with Mowiol on cover glasses (Matsunami, 24 × 60 mm, 0.12–0.17 mm). Immunofluorescent images were captured and analyzed using a LSM710 Zeiss confocal microscopy.