Cool snap es2

Dorsal root ganglion neurons were loaded for 30 minutes at 37°C with 3 μM Fura-2AM (Cat# F1221, Thermo Fisher, stock solution prepared at 1mM in DMSO, 0.02% pluronic acid, Cat#P-3000MP, Life Technologies) to follow changes in intracellular calcium([Ca²⁺]_c) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM Na HEPES, pH 7.4, 5 mM glucose exactly as previously described [8 ]. Fluorescence imaging was performed with an inverted microscope, NikonEclipseTi-U (Nikon Instruments Inc., Melville, NY), using objective Nikon Fluor 4X and a Photometrics cooled CCD camera Cool SNAP ES² (Roper Scientific, Tucson, AZ) controlled by Nis Elements software (version 4.20, Nikon Instruments). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340 ± 5 and 380 ± 7) were controlled by a Lambda 10 to 2 optical filter change (Sutter Instruments). Fluorescence was recorded through a 505-nm dichroic mirror at 535 ± 25 nm. To minimize photobleaching and phototoxicity, the images were taken every ~10 seconds during the time-course of the experiment using the minimal exposure time that provided acceptable image quality. The changes in [Ca²⁺]_c were monitored by following a ratio of F₃₄₀/F₃₈₀, calculated after subtracting the background from both channels.

Dorsal root ganglion neurons were loaded for 30 minutes at 37˚C with 3 µM Fura-2AM (Cat# F1221, Thermo Fisher, stock solution prepared at 1mM in DMSO, 0.02% pluronic acid, Cat#P-3000MP, Life Technologies) to follow changes in intracellular calcium([Ca²⁺]_c) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM Na HEPES, pH 7.4, 5 mM glucose, exactly as previously described [9 ]. Fluorescence imaging was performed with an inverted microscope, NikonEclipseTi-U (Nikon Instruments Inc., Melville, NY), using objective Nikon Fluor 4X and a Photometrics cooled CCD camera Cool SNAP ES² (Roper Scientific, Tucson, AZ) controlled by Nis Elements software (version 4.20, Nikon Instruments). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340 ± 5 nm and 380 ± 7 nm) were controlled by a Lambda 10 to 2 optical filter change (Sutter Instruments). Fluorescence was recorded through a 505-nm dichroic mirror at 535 ± 25 nm. To minimize photobleaching and phototoxicity, the images were taken every ~10 seconds during the time-course of the experiment using the minimal exposure time that provided acceptable image quality. The changes in [Ca²⁺]_c were monitored by following a ratio of F₃₄₀/F₃₈₀, calculated after subtracting the background from both channels.

DRG neurons in culture were treated with either 20 μM of t-CSM or 0.1% DMSO overnight. The neurons were washed in ice-cold PBS twice before incubation (40 min) with anti-NaV1.7 antibody (extracellular epitope, dilution 1/40, Cat# ASC-027, Alomone labs, Israel) for ice. Cells were washed in ice-cold PBS thrice before fixation with 4% paraformaldehyde (10 min, 4°C). The neurons were next kept in blocking buffer (BSA (3% w/v) in PBS) for 20 min at room temperature (RT) before addition of the (goat anti rabbit Alexa Fluor 488 secondary antibody (Catalogue # A-11008, Thermofisher) for 1 hour at RT. After 3 washes with PBS, the coverslips were fixed and imaged with a Nikon Eclipse Ti-U (Nikon instruments) using a Photometrics cooled CCD camera CoolSNAP^ES2 (Roper Scientific, Tucson, AZ) run by Nis Elements software (version 4.20, Nikon Instruments). The fluorescence intensity for each cell was quantified and normalized to the area of the region of interest using Nis Elements software. To obtain the region of interest, the perimeter of each neuron was manually traced.