Sulforhodamine b

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS) and Trypsin were purchased from Gibco-Invitrogen (Grand Island, NY, USA). Dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktail (leupeptin, antipain, chymostatin, and pepstatin A), sulforhodamine B (SRB), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), D(-)-Fructose, Nile Red, Hoechst 33342, tetramethylrhodamine methyl ester (TMRM), and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) were purchased from Invitrogen (Eugene, OR, USA).

We analyzed the diffusion of the gap-junction channel-permeable dyes LY (see above) and sulforhodamine-B, dialyzed in whole-cell configuration in current clamp (zero current) mode. The patch pipettes were filled with ICS described above and containing 2 mM of LY or sulforhodamine-B. LY (Sigma-Aldrich, MW 457.25), is a green- to yellow-fluorescent negatively charged (-2 charges) dye, while sulforhodamine-B (Molecular Probes, MW 558.66) is an orange- to red-fluorescent non-charged water-soluble (polar) sulfonic acid tracer with strong absorption and good photostability [63] (link), [72] (link), [73] (link), [74] (link), [75] (link).

Exocytosis was visualized by using a fluid-phase tracer, sulforhodamine B (0.7 mM; Molecular Probes, Carlsbad, CA, USA) in an assay solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES-NaOH) containing 2.8 mM (low) or 20 mM (high) glucose with two-photon excitation imaging. The islets were imaged with an inverted microscope (IX70; Olympus, Tokyo, Japan) and a laser-scanning microscope (FluoView; Olympus) equipped with a water-immersion objective lens (UplanApo60xW/IR; NA, 1.2), as previously described (Kasai et al., 2005 (link)). Two-photon excitation was performed at 830 nm, and the fluorescence signals of the Ca²⁺ indicator fura-2 (Kd: ∼200 nM) and the polar tracer sulforhodamine B were separated by a dichroic filter and captured at 420–560 and 570–650 nm, respectively, and images were acquired every 1 sec. In an individual, 4,500–5,500 µm² region of interest in islets containing approximately 40–60 cells, we analyzed abruptly appearing small fluorescent spots on the plasma membrane, which were recorded as “exocytic events”.

To identify elastic microfibrils, elastin staining was performed using a Verhoeff van Gieson Elastic Stain Kit (Cat# HT25A, Sigma) in whole eye cross-sections according to the manufacturer’s protocol. Images were captured and analyzed (KEYENCE microscope).
For two-photon imaging, ex vivo mouse eyes were labeled intact, without dissection or sectioning, with Sulforhodamine-B (Thermo Fisher Scientific), a water soluble stain of elastic microfibrils. Microscopy was performed on a Leica SP5 microscope (Leica Microsystems, Heidelberg, Germany) coupled to a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA) through inverted 20X/0.7NA or 63X/1.3NA objectives. We used 850 nm excitation, pulsed, and focused through green (525/50 nm) or red (585/40 nm) filters (Chroma, Bellows Falls, VT) onto a non-descanned photomultiplier tube detector (Hamamatsu, Bridgewater, NJ). Images were collected as multiple channel z-stacks using 512 × 512 or 1024 × 1024 pixel frames, 16-bit grayscale resolution, and 16X line averaging using 1–8 μm step sizes. Images were viewed and processed on LAS AF and AF Lite 2.2.1 (Leica), Image J (NIH; Bethesda, MD), Photoshop CS5 (Adobe, San Jose, CA) and Imaris (Oxford Instruments). Methods reported here have previously been described^{101 (link), 106 (link)–108 (link)}.

H-Cys-2-ClTrt, Fmoc amino acids and amide coupling reagents were purchased from Novabiochem (La Jolla, CA). Solvents were obtained from Sigma Aldrich (St. Louis, MO). RPMI and phosphate buffered saline (PBS) were purchased from Invitrogen (Eugene, OR). Bodipy FL NHS ester reagent, sulforhodamine B, and all other reagents were acquired from ThermoFischer Scientific (Waltham, MA).