Photostable Cy3 Fluorophore Labeling Protocol

A 10 mM pH 7 phosphate buffered saline (PBS, NaH₂PO₄•H₂O: Na₂HPO₄ = 1:3.4 without additional salt) and a 10 mM pH 5 sodium acetate buffer were prepared and filtered through a disposable Millex-GP syringe filter unit (0.22 μm) before use. Glass cover slips (20 mm × 20 mm square, Karl Hecht, Sondheim, Germany); potassium dichromate (ICN Biomedicals Inc. Aurora, Ohio); deionized (DI) water (18.2 MΩ, 0.22 μm pore size filter, APS Water Services Corp., Van Nuys, CA) were used for all experiments. A 50 mM 1-(3-aminopropyl) silatrane (APS) stock solution was prepared by dissolving the APS powder in DI water. The intra-molecularly photostabilized Cy3 fluorophore was chemically synthesized^{20 (link)}. Maleimide-poly (ethylene glycol)-succinimidyl valerate (Mal-PEG-SVA; 3.4 kDa) and methoxy-poly (ethylene glycol)-succinimidyl valerate (mPEG-SVA, 2 KDa) were purchased from Laysan Bio Inc, Arab, AL. Additional reagents included: sodium bicarbonate (Mallinckrodt Baker Inc, Paris, Kentucky); sodium hydroxide (GR grade, EM science, Darmstadt, Germany); sodium phosphate monobasic (ACS grade, Fisher Scientific, Fair Lawn, NJ); sodium phosphate dibasic anhydrous (ACS grade, Fisher Scientific, Fair Lawn, NJ); Amicon 10 kDa and 30 kDa centrifugal filter units (EMD Millipore Co. Billerica, MA); and a 0.1-mm-thick teflon spacer (American Durafilm, Holliston, MA).