Specimens were analyzed using a Nikon C1s confocal laser-scanning microscope, equipped with a Nikon PlanApo 60, 1.4-NA oil immersion lens. Excitation light for fenretinide was obtained by an Argon Ion Laser (405 nm), and emission was recorded at 650 nm. Optical sections were obtained at increments of 0.01 µm in the Z-axis and were digitized with a scanning mode format of 512–512 pixels. Image processing was performed using ImageJ software.
Plan apo 60
Manufactured by Nikon
Sourced in Japan
The Plan Apo 60 is a high-quality optical lens designed for use in various laboratory applications. It features a planar image field and apochromatic correction, providing excellent image quality and resolution. The lens is suitable for a wide range of magnification levels and can be used with a variety of imaging systems.
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Lab products found in correlation
C1s confocal laser scanning microscope, by Nikon (2 mentions)
Eclipse ti, by Nikon (2 mentions)
Alexa fluor 488 and 555, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Neo scmos, by Oxford Instruments (1 mentions)
Gfp hq ex455 485, by Nikon (1 mentions)
H 1000, by Vector Laboratories (1 mentions)
Opera phenix platform, by PerkinElmer (1 mentions)
Ab108986, by Abcam (1 mentions)
A1 confocal laser microscope system, by Nikon (1 mentions)
Diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Plan apo 20, by Nikon (1 mentions)
Spcm aqr 14, by PerkinElmer (1 mentions)
Prime 95b camera, by Nikon (1 mentions)
Ti2 e stand, by Nikon (1 mentions)
Plan fluor 20, by Nikon (1 mentions)
Plan fluor 10, by Nikon (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Te2000 microscope, by Nikon (1 mentions)
17 protocols using plan apo 60
1
Confocal Microscopy of Fenretinide
2
Visualization of DSC1 and BubR1 in siRNA-transfected HaCaT cells
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siRNA-transfected HaCaT cells were rinsed in PBS at 37 °C, fixed in cold methanol for 3 min at −20 °C, blocked for 30 min at room temperature using PBS containing 2% BSA and 2% normal goat serum, and incubated with the following antibodies at the indicated dilutions: rat anti-DSC1 at 1:100 (MAB7367; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-BubR1 at 1:100 (612503; BD Biosciences, San Jose, CA, USA). Cells were rinsed in PBS at 37 °C, fixed in 4% paraformaldehyde for 15 min at 37 °C, permeabilized for 5 min at room temperature using PBS containing 0.2% Triton X-100, and blocked for 30 min at room temperature using PBS containing 2% BSA and 2% normal goat serum. Secondary antibodies conjugated to Alexa Fluor 488 and 555 (Molecular Probes) were used at 1:2,000 dilution. After being washed in PBS containing 4,6-diamidino-2-phenylindole (DAPI) for 5 min, the coverslips were mounted in ProLong Gold (Thermo Fisher Scientific Inc). Fluorescence image acquisition was performed using a Nikon A1R confocal imaging system controlled by Nikon NIS Elements software (Nikon). The objective lens was an oil immersion Plan-Apo ×60 numerical aperture 1.40 lens (Nikon). Images were acquired as Z-stacks at 0.2-μm intervals, and maximum-intensity projections were generated using NIS Elements.
3
Fluorescence Microscopy Imaging Protocol
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Samples were illuminated with a 100 W mercury lamp and visualized by using an epi-fluorescence microscope (Eclipse Ti; Nikon) using an oil-coupled Plan Apo 60 × 1.40 objective (Nikon). Filter blocks with UV-cut specification (TRITC: EX540/25, DM565, BA606/55; GFP-HQ: EX455-485, DM495, BA500-545; Nikon) were used in the optical path of the microscope that allowed the visualization of samples but eliminated the UV part of radiation and minimized the harmful effect of UV radiation on samples. Images were captured using a cooled CMOS camera (Neo sCMOS; Andor) connected to a PC. To capture a field of view for more than several minutes, ND filters (ND4, 25% transmittance) were inserted into the illuminating light path of the fluorescence microscope to avoid photobleaching.
4
Visualization and Quantification of Lipid Droplets in ARPE19 Cells
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Cells were grown on glass coverslips and cultured/treated as described above. ARPE19 mito‐QC cells were fixed in 3.7% PFA at pH 7.0 in 0.2 M HEPES, counterstained with Hoechst 33342 and VECTASHIELD Antifade Mounting Medium H‐1000 was used to mount coverslips on slides. Images were acquired using an ANDOR Spinning Disc Microscope equipped with a Zyla camera (Plan Apochromat ×40 objective, NA 1.15). For high‐content temporal analysis of LD biogenesis, ARPE19 cells were seeded on Ibidi black wall m plates (24 well format) with culture and treatment conditions as described above. Samples were labelled with BODIPY and MitoTracker and fixed as described above, followed by imaging with a PerkinElmer Opera Phenix Platform. For analysis of LDs in ULK1 KO cells, images were acquired using a wide‐field Nikon Eclipse Ti wide‐field microscope using a Nikon Plan Apo ×60 oil immersion objective.
5
Microscopy Imaging Protocol for Cellular Visualisation
6
Immunohistochemical Analysis of Prostate Samples
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At the endpoint of CP1 instillation experiment, the mice were euthanized, and the prostates were harvested from mice as described previously (34 (link)). Prostate samples were fixed in 10% formalin, processed, and embedded in paraffin by the Northwestern University Mouse Histology and Phenotyping Core facility. The formalin-fixed paraffin-embedded (FFPE) samples were then sectioned (5 μm sections) and mounted on glass slides for staining.
The antibodies used in this study were as follows: rabbit anti-PGP9.5 (1:500, catalog # ab108986, RRID: AB_10891773; abcam, Cambridge, UK) and mouse anti-human mast cell tryptase (1:500, with cross-reactivity with mouse, catalog # 369402, RRID: AB_2566541; BioLegend®, San Diego, CA). The immunolabeling was visualized by using Cytm2-conjugated donkey anti-mouse IgG and Cytm5-conjugated-donkey anti-rabbit IgG and mounted using diamond Antifade mounting medium (Invitrogen, Thermo Fisher, Hampton, NH).
Images were obtained on a Nikon A1 Confocal Laser Microscope System (Plan Apo 20 × NA 0.75 and Plan Apo 60 × NA1.4 Oil, objectives). Images were taken on the same confocal imaging settings as the template for acquiring all images.
The antibodies used in this study were as follows: rabbit anti-PGP9.5 (1:500, catalog # ab108986, RRID: AB_10891773; abcam, Cambridge, UK) and mouse anti-human mast cell tryptase (1:500, with cross-reactivity with mouse, catalog # 369402, RRID: AB_2566541; BioLegend®, San Diego, CA). The immunolabeling was visualized by using Cytm2-conjugated donkey anti-mouse IgG and Cytm5-conjugated-donkey anti-rabbit IgG and mounted using diamond Antifade mounting medium (Invitrogen, Thermo Fisher, Hampton, NH).
Images were obtained on a Nikon A1 Confocal Laser Microscope System (Plan Apo 20 × NA 0.75 and Plan Apo 60 × NA1.4 Oil, objectives). Images were taken on the same confocal imaging settings as the template for acquiring all images.
7
Single-Molecule FRET Microscopy Assay
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A home-built dual-channel confocal fluorescence microscope was used to detect freely diffusing single molecules of dual-labelled RNA aptamers. Apparent FRET efficiencies, Eapp, of each burst were calculated according to Eapp = nA/(nA + nD), where nA and nD are the acceptor and donor counts, respectively.44 (link) The donor, fluorescence, was excited by an argon ion laser (model 35LAP321-240, Melles Griot) with 150 μW at 488 nm. Donor and acceptor fluorescence was collected through an oil-immersion objective (Nikon PlanApo ×60, numerical aperture 1.45) and detected separately by two photon-counting modules (SPCMAQR14, PerkinElmer). The outputs of the two detectors were recorded by two computer-implemented multichannel scalar cards (MCS-PCI, ORTEC). A threshold of 25 counts per millisecond bin for the sum of the donor and acceptor fluorescence signals was used to differentiate single molecule bursts from the background.
8
Live Imaging of Drosophila Embryogenesis
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Flies were raised at 25°C under standard conditions. Pupae were collected for imaging as described previously [52 ]. Ecad::GFP flies [53 (link)] were used for live imaging as previously described [1 (link)]. In brief, images were acquired with a spinning disk microscope from Gataca Systems driven by the MetaMorph software. The system is equipped with an inverted Nikon TI2E stand, a motorized XYZ stage, and a Nikon Plan Apo ×60 oil immersion (NA=1.4) lens and with a Prime95B camera.
9
Confocal Microscopy Imaging of Fluorescently Labeled Proteins
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Images were obtained at room temperature by using a Perkin-Elmer Ultraview LTE Confocal microscope with a Nikon TE-2000 microscope. For fluorescence imaging, a Nikon Plan Apo ×60 oil immersion lens with the numerical aperture (NA) 1.4 was used. For brightfield imaging, a Nikon Plan Fluor ×10 dry lens (NA = 0.3) or Nikon Plan Fluor ×20 dry lens (NA = 0.45) was used. Proteins were labeled using the fluorochromes Alexa 488, Alexa 568 or mRFP-1, as described above. The images were acquired using an Hamamatsu ORCA ER or EMCCD C9100-02 camera and the Volocity Acquisition module (Improvision, PerkinElmer) software.
10
Fluorescence Correlation Spectroscopy in Spheroids
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Fluorescence correlation spectroscopy experiments were performed on a dedicated FCS system, based on a Nikon C1 inverted confocal microscope (Nikon Instruments, Japan) with a PlanApo 60×, NA = 1.2 water immersion objective. The setup is equipped with a Pico Harp 300 system (PicoQuant, Germany). The body of the microscope was enclosed in a climate chamber (Okolab, Italy), providing a temperature control at 36.0 ± 0.5 °C and the required humidity. All the tracers were excited using a 561 nm laser, and the fluorescence was detected through a 593/46 bandpass filter (Chroma, USA). Data acquisition was controlled using the SymPhoTime 64 software (PicoQuant, Germany). The experiments were preceded by establishing the dimension of the confocal volume using rhodamine B (Sigma-Aldrich, USA) dissolved in 2.5% glucose in PBS. 18 FCS measurements were performed at a depth of 10-30 µm within the spheroids (Fig. 1), in the extracellular space at the distance of 2-10 µm from the cell edges. The detection volume was positioned in the ECM using the imaging mode of the microscope (ESI, Fig. S7 †).
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