Observer z1 microscope

To examine the localization of the various GFP tagged pleckstrin homology (PH) domains, PC12 cells were grown on poly-L-Lysine coated cover slides and fixed with 3.7% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA). The fixed cells were imaged using a Zeiss Observer.Z1 microscope with an attached Orca II ERG camera (Hamamatsu, Bridgewater, NJ, USA). Zeiss Axiovision 4.8 software and ZEN 2012 blue edition software were used to acquire images. GFP localization was counted from cells exhibiting a RFP signal, indicating the presence of the shRNA vector. For quantitation of GFP fluorescence, images were analyzed with Image J software (NIH). For measurement of plasma membrane fluorescence, a line was drawn along the arc of the plasma membrane and the average pixel intensity along the line was used as the fluorescence value. For measurement of Golgi fluorescence, an outline was drawn around the Golgi elements and the mean intensity for all pixels within that area was used. Background fluorescence was subtracted from all images prior to measurement using the automated function within Image J.

RoGFP was expressed in primary fibroblasts using modified pEGFP-N1 (RRID:Addgene_38120) as the expression vector and JetPei as the transfection reagent. After the cells were incubated in culture medium treated with or without HU or APH for 72 h at 37 °C, the cells were washed twice with Hanks’ balanced salt solution. For pEGFP-N1/roGFP1, the cells were imaged on a Zeiss Observer Z1 microscope with a Hamamatsu ORCA Flash 4LT camera. Images were acquired using MetaMorph software (Molecular Devices). For dual excitation ratio imaging, excitation filters at wavelengths of 400 nm and 488 nm were used, and an emission filter at a wavelength of 535 nm was used. The fluorescence excitation ratio was obtained by dividing the intensities of the cells using excitation filters at 400 nm and 488 nm.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.