Orca 2 er

Immunofluorescence on cryosections was performed as described (47 (link)) and examined with confocal TCS SP5 laser-scanning confocal (Leica) or Olympus BX (Olympus Optical) fluorescent microscope, and Zeiss Axiovert S100 TV2 with Hamamatsu OrcaII-ER. For immunohistochemistry, sciatic nerves were removed and rapidly snap-frozen in liquid nitrogen, either unfixed or previously fixed in buffered 4% PFA.

For the proliferation assay, C2C12 cells were plated at the density of 2500 cells/cm². The day after, the transient transfection with siRNAs was performed as described above. Cells were counted every 9 or 15 h until 100% of confluence was reached. Doubling times were evaluated as follow: DT = (duration)Log(2))/((Log(final concentration) − Log(initial concentration)).
For the migration assay, C2C12 cells were transfected with siRNAs as described above. At the density of 60%, images were recorded every 5 min for 4 h, using Zeiss Axiovert S100 with Hamamatsu OrcaII-ER. Three fields with ×10 magnification were analyzed for each condition, and 11 mononucleated cells were tracked for each field. The mean of the total distance covered by each cell of the field were evaluated and converted from pixel to μm. The results are expressed as mean of three independent experiments, for a total of 99 cells per condition.

GH3/prolactin-luc cells were seeded in 35-mm glass coverslip-based dishes (IWAKI, Japan) 20 h prior to imaging. Luciferin (1 mM) was added at least 10 h before the start of the experiment, and the cells were transferred to the stage of a Zeiss Axiovert 200 equipped with an XL incubator (maintained at 37°C, 5% CO₂, in humid conditions) maintained within a darkened room. Cells were loaded with Fluo-4 for 30 min and then time-series imaging was performed using a Fluar x20, 0.75 NA (Zeiss) air objective, with an argon-ion laser at 488 nm. Emitted light was captured through a 505–550 nm bandpass filter from a 545 nm dichroic mirror. Calcium recordings were captured every 1 s for at least 250 s unless stated otherwise. Data were captured using LSM510 software with consecutive autofocus. The microscope and all light emitting devices were then shut down and luminescence images were captured using a photon-counting charge coupled device camera (Orca II ER, Hamamatsu Photonics, UK). Sequential images, integrated over 30 min, were taken using 4 × 4 binning and acquired using Kinetic Imaging software AQM6 (Andor, UK). Bright field images were taken before and after luminescence imaging to allow localization of cells. In the relevant experiments, 0.5 µM BayK8644 was added to the dish at around 100 s during the calcium imaging period.

For time-course imaging of competition experiments, cultures were set up as described above, with the addition of IPTG to the starting overnight culture and to the competition flasks to induce fluorescent reporter expression. At each time point, cells were harvested, diluted in one volume PYE and propidium iodide added to a final concentration of 15 µM. Cells were incubated in the dark for 15 min, spotted onto PYE 1.5% agarose pads and imaged immediately. Phase contrast and fluorescent images were taken on a Zeiss Axiovert 200M microscope with a 63× phase or αFluar 100×/1.45 oil immersion objective, using a digital camera (Orca-II ER; Hamamatsu Photonics) and Metamorph software (Universal Imaging, PA). The following emission/excitation filters were used: YFP, 500/25 and 535/30m; CFP, 436/25 and 480/40; mCherry, 560/40 and 630/75. Image analysis to quantify fluorescent signals for each cell was calculated using MicrobeJ (Ducret et al., 2016 (link)). Three independent replicates were analyzed, with a minimum of 250 cells per frame. Fold induction for the P_cdzC-YFP transcriptional reporter was calculated relative to fluorescence density at OD₆₀₀ = 0.025. Image overlays were generated using Fiji (Schindelin et al., 2012 (link)).

Briefly, for immunocytochemical analyses, cell cultures were rinsed in PBS and fixed for 15 min in 4% paraformaldehyde/PBS. Coverslips were then washed three times in PBS and incubated for 30 min in blocking solution (2% goat serum, 2% serum albumin, 0.1% Triton X-100 in PBS). Antibodies were diluted to the appropriate concentration in blocking solution. Coverslips were incubated for 60 min in the antibody solution. The samples were subsequently washed three times and incubated for 30 min with the appropriate fluorescence-conjugated secondary antibodies. Finally, coverslips were washed five times and mounted with Mowiol. Fluorescence images were obtained with an inverted microscope (Olympus IX70), using a TILL monochromator as light source. Pictures were taken with an attached cooled CCD camera (Orca II-ER, Hamamatsu).
The following antibodies were used: anti-vinculin monoclonal antibody (Chemicon, ref. MAB3574) and anti-Profilin polyclonal and monoclonal antibodies (Cell Signaling, ref. 3237 and Synaptic system, ref. 308 011). Secondary antibodies, Oregon green Phalloidin and Phalloidin-Alexa Fluor® 594 were purchased from Invitrogen. Image J analysis software (National Institutes of Health) was used to quantify spreading and focal adhesion.