Cell a imaging software

One day before the transfection, Neuro-2a cells were applied to coverslips and placed inside 35-mm cell culture dishes with 2 ml of Eagle’s medium. Transfection medium contained cDNA of human α7 nAChR (3 mg/ml) and the Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific). The cDNA-containing solution was replaced by cell culture medium 6–8 h after the transfection.
Cryostat 10 μm-thick sections of rat tongue were fixed with isopropanol for 10 min at 4°C, rinsed with PBS and distilled water, air dried for 1 h (all further procedures were carried out at room temperature) and incubated for 1 h with PBS, pH 7.4, containing 10 mg/ml BSA and 5 ml/L Tween 20 to block unspecific binding of toxins. The sections were pre-incubated for 1–2 h in buffer A (1 mg/ml BSA and 150 mM NaCl in PBS). Controls were run simultaneously by adding 300-fold excess of unlabeled α-CbTx (unless specified otherwise). Then, AF555–α-BgTx was added to the slides to reach 50 nM final concentration and they were incubated for 1–15 h. Sections were subsequently washed with PBS, fixed with 40 mg/ml paraformaldehyde for 10 min, rinsed with PBS again and coverslipped in carbonate-buffered glycerol at pH 8.6. The slides were analyzed using CellA Imaging Software (Olympus Soft Imaging Solutions, Germany) coupled to epifluorescent microscope with cooled CCD CAM-XM10 (Olympus, Japan).

For measurements of intracellular calcium concentration [Ca²⁺]_i changes, transfected Neuro2a cells plated on glass coverslips were perfused at room temperature with the buffer containing 140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4. Expression of Case12, a fluorescent genetically encoded sensor of calcium ions (ex/em = 491/516 nm), allowed direct monitoring of changes in [Ca²⁺]_i using an epifluorescence microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Japan). Videos were made and processed using CellA Imaging Software (Olympus Soft Imaging Solutions GmbH, Germany), Image J, CellX, and OriginPro 7.5 software (OriginLab, MA, USA, for statistical analysis). The cells were exposed to acetylcholine iodide (Sigma, Germany), epibatidine (Tocris, UK), and α-cobratoxin purified from Naja kaouthia venom, and changes in Case12 fluorescence were recorded from each cell independently. To increase the registered changes, all ligand solutions contained the α7 nAChR positive allosteric modulator PNU120596 (10 μM, Tocris, UK). To allow recovery of the cells, the washing steps lasted 5–10 minutes. All recordings were made at room temperature.