Cover glass

Trophoblast invasion ability was determined using a 24-well cell culture transwell system with pore size of 8 μm (Falcon, BD biosciences, Franklin Lakes, NJ, USA). After HTR-8/SVneo cells (3 × 10⁴) were added to the upper chamber with 0.3 mL serum-free medium whereas culture medium containing 5% FBS was added to the lower chamber, they were incubated for 24 h. To measure the number of invaded cells, insert membranes were fixed using 4× paraformaldehyde (PFA; elbio, Seoul, Korea) and 100% methanol (Ducksan, Seoul, Korea) and stained with Mayer’s Hematoxylin (Dako, Carpinteria, CA, USA). After washing with phosphate-buffered saline (PBS), stained membrane of transwell was mounted onto a slide glass (Marienfeld, Lauda-Königshofen, Germany) using mounting solution (Dako, Carpinteria, CA, USA) and fixed with a cover glass (Marienfeld, Lauda-Königshofen, Germany). Numbers of stained cells in at least nine randomly selected nonoverlapping fields were counted under a light microscope. All experiments were performed in triplicate.

Sections were mounted on glass slides (Superfrost Plus, Thermo Scientific) and washed three times in 95% ethanol for 2 min, followed by incubation in xylene for 5 min. Slides were washed in 100%, 95% ethanol, and water for 5 min and 2 min, respectively, followed by staining in Nissl solution for 5 min. Stained sections were washed three times for 2 min in water and incubated in a formalin acetic solution for 10 min. Three washes in water for 2 min and two washes in 100% ethanol for 5 min were conducted followed by fixing twice in xylene for 10 min. Sections were mounted with DPX mountant (Sigma) and covered with a coverglass (Marienfeld). Stained images were acquired using a Leica microscope (CTR6000, Leica, Germany). Total numbers of Nissl-stained Purkinje cells in cerebellar lobules were counted using Image J software (NIH, USA). For Nissl counting, a cell was defined as a bright blue-stained nucleolus^{4 (link)}. Counted cells were quantified from at least 3 independent experiments.

For immunofluorescence microscopy, Hepa1-6 cells were seeded on a cover glass (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) in a 12-well plate, fixed with 4% formaldehyde, and subsequently permeabilized using 0.01% Triton X-100. After blocking with 2% FBS for 30 min, the cells were incubated with anti-HIF-1α (Novus Biologicals) or anti-γH2AX (Cell Signaling Technology) primary antibodies, or with Alexa-Fluor488-conjugated phalloidin (Life Technologies, Eugene, OR, USA) for 1 h; next, the cells were incubated with DAPI (Sigma-Aldrich) and Alexa-Fluor-488-conjugated secondary antibodies for 30 min. Anti-rabbit and anti-mouse Alexa-Fluor-488-conjugated secondary antibodies were obtained from Molecular Probes (Carlsbad, CA, USA). The cells were washed, mounted using a fluorescent mounting medium (Dako, Carpinteria, CA, USA), and analyzed with fluorescent microscopy (Zeiss Observer D1; Carl Zeiss Co., Ltd., Seoul, Korea).

MDCK cell islands at 3, 6, 9 and 12 hours after releasing were pre-treated with 0.4% Triton X-100/0.4% paraformaldehyde in PBS for 3 min and fixed with 4% paraformaldehyde in PBS for 20 min. Cells were permeabilized with 0.4% Triton X-100 and blocked with 5% bovine serum albumin (BSA). The antibody for E-cadherin (24E10; 1:50; Cell Signaling) or Vinculin (hVIN-1; 1:400; Sigma-Aldrich) was treated to the cell islands. After washing the cells with PBST, Texas Red-conjugated secondary antibody, Alexa Fluor 488 phalloidin (1:200; Life Technologies) and Hoechst (33342; 1:3000; Life Technologies) were added for staining the cell islands. Cells were mounted in Elvanol reagent and covered with a cover glass (diameter = 12 mm; Marienfeld). Fluorescent images were taken with using a fluorescence microscope (Axioscope; Carl Zeiss) and a confocal microscope (LSM 510, Carl Zeiss). The confocal images were captured ~15 images at 100 nm interval per a position. In order to observe representative regions for focal adhesion and adherens junction, 4 images at the basal sections (0.1~0.4 μm) and in the mid-apical sections (0.9~1.2 μm) were respectively merged by projections of maximal intensity using ImageJ.