Silicone-Urethane Adhesive for Improved Coverslip Mounting and Leak-Free Preparation Of

Coverslip treatment for silicone-urethane adhesive mounting

PROTOCOL FOR: Silicone-urethane adhesive for improved coverslip mounting and leak- free preparation of living cell observation chambers

Shigeru Matsunaga, Qiuhong Xie, Miyuki Kumano-Kuramochi, Shiro Komba, and Sachiko Machida

National Food Research Institute, Tsukuba, Ibaraki, Japan

LEGEND

ATTENTION * HINT

REST

REAGENTS  Silicone: Siliconize L-25 (alcohol-based dimethyl polysiloxane solution, Fuji System, Tokyo, Japan) is most convenient for direct use. Silicone is slightly more expensive than VALAP or nail polish, but 1 ampoule of silicone solution (∼$8 USD) is enough to prepare several hundred coverslips. Alternatively, silicone oil DC 200 (Sigma-Aldrich, St. Louis, Missouri, USA) can be used.  Urethane resin: 20–30% solvent-based polyurethane adhesive (where the solvent is ethyl acetate) is suitable [e.g., Seal Primer 7N (Konishi, Osaka, Japan)].  Coverslip: 24 mm × 24 mm, thickness 0.12–0.17 mm (Catalog no. 1; Matsunami, Kishiwada, Japan)  Glass slide: 76 mm × 26 mm, thickness 0.8–1.0 mm, (Catalog no. S1111; Matsunami, Kishiwada, Japan)  Adhesive tape, 25.4-mm width (Scotch 3M, St. Paul, Minnesota, USA)

PROCEDURE

COVERSLIP PREPARATION 1. Dilute Siliconize L-25 with distilled water to 1/50. 2. Immerse an edge of the coverslip 5 mm into diluted Siliconize L-25 and hold for 10 s. 3. Immerse the siliconized edge of the coverslip in distilled water to rinse (this step promotes drying of the silicone) and wipe away the excess liquid with a Kimwipe. 4. Repeat steps 2–3 for each edge of the coverslip. 5. Sterilize the coated coverslips by dry heat (160°C for 2 h). PRETREATMENT OF GLASS SLIDE 6. Place adhesive tape on one side of the glass slide. Paint urethane resin on the tape with a small paintbrush. The urethane resin solution dries quickly. 7. Cut an 18-mm square from the center of tape with a utility knife in order to form a space for the sample between the slide and coverslip.

MOUNTING THE COVERSLIP 8. Grow cells on coated coverslips in culture dishes. *Alternatively, if it is desirable to use minimal amounts of reagents, apply a 0.3-ml drop of cell suspension on the center of a coated coverslip and grow cells. Subject cells to the desired experimental conditions (e.g. treat with various reagents) on the coverslip. 9. Put 50 μl of prewarmed (37°C) medium on the glass slide in the space formed by the tape/urethane boundaries. *Although not necessary, it is useful to prewarm the glass slide to 37°C with a hotplate. This step helps keep the cells healthy during mounting. 10. Remove the coverslip from the culture dish and blot excess medium on a Kimwipe. 11. Invert the coverslip on the glass slide, taking care not to introduce air bubbles. 12. Carefully press the edge of coverslips with a cotton swab to form a good seal. 13. Remove excess solution with a cotton swab or Kimwipe. *This protocol may also be used with nonadherent cells, in which case the sample solution is added in the reservoir created by the tape/urethane boundaries and the silicone-coated coverslip is placed on top.

EQUIPMENT  Paintbrush (Catalog no. FDP 5-3; Pentel, Tokyo, Japan)  Hotplate (Model no. PC-400D; TAITEC, Koshigaya, Japan)  Kimwipes (Wiper S200; Kimberly-Clark, San Antonio, TX, USA)  Oven (Catalog no. OTW-502M; Hirayama, Kasukabe, Japan)  Utility knife (Art Knife 10BS; OLFA, Osaka, Japan)