Immunofluorescence Microscopy of Infected Cells

Approximately 95% confluent infected NMuMG monolayers were analyzed by immunofluorescence 36-48 h post-infection or 48 h post-siRNA transfection as described [34 (link)]. Cells were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI) to visualize the nuclei. A Zeiss Axioplan 2 immunofluorescence microscope was used with the Zeiss 20×, 40× or 63× objective lens and photographing at ambient temperature in the presence of immersion oil. Images were acquired with a Hamamatsu C4742-95 CCD digital camera and the acquisition software QED Camera Plugin v1.1.6 (QED Imaging Inc.) and Volocity^® (PerkinElmer). Images taken were processed with Adobe Photoshop 6.0 or CS2 to reduce file size.

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Vanlandewijck M., Dadras M.S., Lomnytska M., Mahzabin T., Lee Miller M., Busch C., Brunak S., Heldin C.H, & Moustakas A. (2017). The protein kinase SIK downregulates the polarity protein Par3. Oncotarget, 9(5), 5716-5735.

Publication 2017

Axioplan 2 immunofluorescence microscope Volocity®

Camera Cells Immersion Immunofluorescence Immunofluorescence microscope Infection Lens Nuclei Sirna Transfection

Corresponding Organization : Science for Life Laboratory

Other organizations : Integrated Cardio Metabolic Centre, Karolinska Institutet, Uppsala University Hospital, University of Western Australia, Novo Nordisk Foundation, University of Copenhagen, Cancer Research UK Cambridge Center, University of Cambridge

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Variable analysis

independent variables

Infection of NMuMG monolayers
SiRNA transfection of NMuMG cells

dependent variables

Immunofluorescence analysis of NMuMG cells
Visualization of nuclei using DAPI staining

control variables

Approximately 95% confluent NMuMG monolayers
Microscopy imaging using Zeiss Axioplan 2 with 20×, 40×, or 63× objective lens
Imaging at ambient temperature in the presence of immersion oil
Image acquisition using Hamamatsu C4742-95 CCD digital camera and QED Camera Plugin v1.1.6 (QED Imaging Inc.) and Volocity® (PerkinElmer) software
Image processing using Adobe Photoshop 6.0 or CS2 to reduce file size

controls

No positive or negative controls were explicitly mentioned in the provided information.

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