Lsm710 laser scanning meta confocal microscope

To look at the localisation of NGF in undifferentiated cells, 48-h post-transfection, PC12 cells were fixed in 4% paraformaldehyde for 10 min. To look at NGF localisation in differentiated cells, 24 h post-transfection cells were treated with 100 ng/ml mouse NGF (Peprotech) for five days and then fixed. Cells were stained with an mCherry antibody (ThermoFisher Scientific 16D7, 1:1000), and Alexa-546 conjugated secondary antibodies were purchased from Invitrogen (1:1000).
All images were acquired with an LSM710 laser-scanning Meta confocal microscope (Carl Zeiss) using a ×63 oil-immersion objective.

Twenty‐four hours post‐transfection, HEK‐293 cells were washed in PBS and fixed in ice‐cold methanol. Cells were stained with GFP antibody (Abcam, Cambridge, UK; ab6556, 1:1000) and Na⁺/K⁺ ATPase α1 subunit antibody (Abcam; ab7671, 1:300). Alexa‐488 and Alexa‐546 conjugated secondary antibodies were purchased from Invitrogen, Carlsbad, CA, USA (1:1,000). All images were acquired with an LSM710 laser‐scanning META confocal microscope (Carl Zeiss, Oberkochen, Germany) using a ×63 oil‐immersion objective.
Image analysis was performed using the Volocity image analysis software (PerkinElmer, Waltham, MA, USA). Colocalization was quantified in terms of Manders M1 coefficient. The proportion of GFP at the membrane was quantified by measuring the GFP at the plasma membrane and dividing by the total GFP fluorescence.

Twenty‐four hours post‐transfection, calcium imaging was performed on HeLa cells using Rhod‐3‐AM calcium imaging kit (Invitrogen) following manufacturer's protocol. Images were acquired with an LSM710 laser scanning META confocal microscope (Carl Zeiss) using a ×20 objective and maximum pinhole aperture of 600 μm. Two line averages were performed for each frame with images taken every 4 sec.
At t = 48 sec, 20 μl of HBSS media was added, at t = 140 sec, 20 μl NGF (final concentration of 100 ng/ml) was added to the cells. The response to NGF was monitored for 10 min, after which at t = 820 sec, 100 ng/ml ionomycin was added. For PLCγ inhibition studies, WT transfected cells were treated with 3 μg/ml of U73122 (Calbiochem, Billerica, MA, USA) for 30 min prior to NGF stimulation.
Image analysis was performed with the Volocity 3D Image Analysis Software using green cells that did not spontaneously fluoresce and had an ionomycin response.