A sequential immunofluorescence staining protocol in two steps was applied on cancer tissue sections from 5 patients with extensive CD68+ and SIRPα+ and 5 tissue sections with extensive CD68+ but low SIRPα+ macrophage stroma infiltration documented in immunohistochemistry single staining. The anti-CD68 and anti-SIRPα antibodies were used at concentrations and conditions used in immunohistochemistry. Details on the protocol applied have been previously published by our group [24 (link)]. The anti-rabbit Biotium-CF568 (1:250; Fremont, CA, USA) and the anti-mouse Biotium-CF488 (1:250; Fremont, CA, USA) secondary antibodies were used for 30 min incubation, for SIRPα and CD68 staining, respectively. The DNA was counterstained with Hoechst 33,342 for 30 min at room temperature. Image acquisition was performed on a customized Andor Revolution Spinning Disk Confocal system (Yokogawa CSU-X1) built around an Olympus IX81 with 10 × 0.30 NA air lens and a digital camera (Andor Ixon+885) (Bioimaging Facility, MBG-DUTH). The system was controlled by Andor IQ3 software. Images were acquired as z-stacks with a z-step of 2 μm, through the entire volume of the cells.
Csu x1
Manufactured by Olympus
The CSU-X1 is a confocal scanning unit designed for use in fluorescence microscopy applications. It features a high-speed spinning disk that enables rapid image acquisition. The unit is compatible with a range of microscope systems and can be integrated into various imaging setups.
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Lab products found in correlation
Metamorph software, by Molecular Devices (2 mentions)
X cite 120led, by Excelitas (2 mentions)
Axio observer z1, by Zeiss (2 mentions)
Lightsheet z 1, by Zeiss (2 mentions)
X cite 120q, by Excelitas (2 mentions)
Orca flash4.0 v3, by Hamamatsu Photonics (2 mentions)
Ld plan neofluar, by Zeiss (2 mentions)
Zen blue 2012 imaging, by Zeiss (2 mentions)
Ixon3 897 emccd, by Olympus (2 mentions)
35 mm glass bottom dishes, by MatTek (2 mentions)
Spinning disk confocal system, by Yokogawa (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Hibernate e, by Transnetyx (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
40 uapo 340 objective lens, by Olympus (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Uapo 340, by Olympus (1 mentions)
Lcv100, by Olympus (1 mentions)
Dp30 camera, by Olympus (1 mentions)
9 protocols using csu x1
1
Multi-Stain Confocal Imaging of Tumor Macrophages
2
Parkin Trafficking in Rat Hippocampal Neurons
3
Visualizing Wound Healing and Cell Migration
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For analysis of wound healing and cell migration by live imaging, we used a LCV100 (Olympus) equipped with a UAPO 40×/340× objective lens (Olympus), an LED light source, a DP30 camera (Olympus), and DIC optical components and interference filters, except Video 2 , for which we used an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and electron-multiplying charge coupled device (EMCCD; iXon+, Andor Technology). To observe actin dynamics, we isolated stable lines of wild-type and αEcat KO Caco-2 cells expressing LifeAct-RFP. A wild-type line of these transfectants was additionally transfected with Nap1-GFP in a transient way. These cells were observed using an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and a 561-nm laser (Sapphire LP, Coherent) for RFP excitation or a 488-nm laser (Sapphire LP, Coherent) for GFP excitation. We took fluorescence images with multiple z-stacks by EMCCD (iXon+, Andor Technology) at the specified time intervals and then made maximum-intensity Z projections.
4
Imaging Granulomas Using Epifluorescence and Confocal Microscopy
5
Real-time imaging of DNA repair dynamics
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Exponentially growing human U2OS cells were plated onto 35-mm glass-bottom dishes (MatTek) and transfected with the pTGFP-H2AZ construct, pEGFP-RUVBL2 (Origene) or pEGFP-H2B using NanoJuice according to the manufacturer’s protocol. The cells were allowed to express the construct for 24 h and were then incubated with 10 μg/ml Hoechst 3458 for 30 min at 37°C before irradiation. The microscope system used was an Intelligent Imaging Innovations spinning disc confocal with a Yokogawa CSU-X1 on an Olympus IX-71. GFP-positive cells were irradiated with 405-nm ultraviolet laser set at power of 7:1,000 for either H2AZ or H2B and at 30:1,000 for RUVBL2 and channelled through a 60× objective. Images were captured at 10-s intervals following laser damage for a total time of 5 min for H2AZ or H2B and 30 min for RUVBL2. Images generated were acquired on a Photometrics Evolve 512 × 512 EMCCD using Slidebook 6 software. In protein recruitment experiments, signal intensity was quantified along the laser path, using Slidebook 6 software, in a minimum of 10 cells and error bars represent the SD between three independent experiments.
6
Spatiotemporal Dynamics of SETDB1 in DNA Damage Response
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U2OS cells were seeded onto 35 mm glass-bottom dishes (MatTek) and transfected with pEGFP-SETDB1 constructs using Gene Juice transfection reagent. Cells were grown for 48 h and, when used, 10 μM ATMi (Tocris Bioscience) added 1 h prior to the exposure to laser microirradiation. Cells were incubated with 10 μg/ml Hoechst 34580 for 30 min at 37°C before exposure. The Intelligent Imaging Innovations spinning disk confocal microscopy with a Yokogawa CSU-X1 on an Olympus IX-71 was used for imaging. EGFP positive cells were irradiated with a 50 mW, 405 nm ultraviolet laser and channelled through a 60× objective. The UV laser was focused to an area of ∼12 × 0.1μm through the cell nuclei, and images were captured at 10 s intervals following laser damage for a total time of 210. Signal intensity was quantified along the laser path using Slidebook 6 software. As controls, cells were exposed to laser microirradiation without Hoescht treatment and no signal was obtained in all cases.
7
Measuring Nuclear-to-Cytoplasmic Ratio using Confocal Imaging
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Confocal imaging utilized a PerkinElmer UltraVIEW VoX with a Yokogawa CSU-X1 spinning disk head, a 100× 1.46 NA Olympus Plan Apo oil objective, and CCD (ORCA-R2) and EMCCD (C9100-13) cameras. All confocal microscopy was performed at room temperature (22–23°C). GFP/mCherry images were taken using a 488-nm laser (for GFP) or a 561-nm laser (for mCherry), with alternating excitation. Images were collected using the Volocity imaging software.
To measure the intensity of NLS-GFP, sum projections of the entire z-stack were created using ImageJ. After background subtraction, a region of interest (ROI) was manually drawn around each nucleus and the integrated fluorescence intensity was divided by the area of the ROI. This ROI was then used to measure the integrated fluorescence intensity of the cytoplasm of that cell. The integrated fluorescence intensity of the nucleus over the cytoplasm gave a N:C ratio for each cell. The average of this ratio for 100 cells was determined.
To measure the intensity of NLS-GFP, sum projections of the entire z-stack were created using ImageJ. After background subtraction, a region of interest (ROI) was manually drawn around each nucleus and the integrated fluorescence intensity was divided by the area of the ROI. This ROI was then used to measure the integrated fluorescence intensity of the cytoplasm of that cell. The integrated fluorescence intensity of the nucleus over the cytoplasm gave a N:C ratio for each cell. The average of this ratio for 100 cells was determined.
8
Imaging Granulomas Using Epifluorescence and Confocal Microscopy
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Epifluorescent imaging of granulomas was conducted on an inverted Zeiss AxioObserverZ.1 using a 20× long working distance/0.4 NA lens (Zeiss, LD Plan NeoFluar), a Zeiss MRm camera and either an Xcite 120Q or an Xcite 120LED (Lumen Dynamics) light source. Images were acquired using the Zen Blue 2012 imaging suite (Zeiss).
Confocal images were acquired on an Andor XD spinning disk confocal, consisting of an Olympus IX81 inverted microscope equipped with a Yokogawa CSU-X1 spinning disk using a 20×/0.5 UplanFl lens (Olympus) and detected with a Andor Ixon3 897 EMCCD. Images were acquired using Metamorph.
Confocal images of CLARITY cleared granulomas and granulomas from (cdh1-tdtomato)xt18 animals were obtained on a Zeiss AxioObserverZ.1 equipped with an XLIGHT V2 spinning disk unit (Crest Optics, Rome, Italy) using a 20×/0.5 NA lens, an ORCA Flash4.0 V3 (Hamamatsu, Bridgewater, NJ) and an LDI multiline laser (89 North, Williston VT).
Lightsheet microscopy was performed on a Zeiss Lightsheet Z1 using a Plan-Apochromat 20×/1.0 NA Aqueous immersion objective and a PCO.edge camera. Individual channels were acquired sequentially using dual-side illumination.
For presentation, images were prepared and adjusted using Zen software and FIJI for contrast adjustments and maximum projections.
Confocal images were acquired on an Andor XD spinning disk confocal, consisting of an Olympus IX81 inverted microscope equipped with a Yokogawa CSU-X1 spinning disk using a 20×/0.5 UplanFl lens (Olympus) and detected with a Andor Ixon3 897 EMCCD. Images were acquired using Metamorph.
Confocal images of CLARITY cleared granulomas and granulomas from (cdh1-tdtomato)xt18 animals were obtained on a Zeiss AxioObserverZ.1 equipped with an XLIGHT V2 spinning disk unit (Crest Optics, Rome, Italy) using a 20×/0.5 NA lens, an ORCA Flash4.0 V3 (Hamamatsu, Bridgewater, NJ) and an LDI multiline laser (89 North, Williston VT).
Lightsheet microscopy was performed on a Zeiss Lightsheet Z1 using a Plan-Apochromat 20×/1.0 NA Aqueous immersion objective and a PCO.edge camera. Individual channels were acquired sequentially using dual-side illumination.
For presentation, images were prepared and adjusted using Zen software and FIJI for contrast adjustments and maximum projections.
9
4D Imaging of C. elegans Embryogenesis
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Embryos were collected from gravid hermaphrodites and mounted with polystyrene beads (Polysciences Inc) as described (Du et al., 2015 (link)). Embryos were imaged on a Zeiss AxioObserver Z1 inverted microscope frame with Yokogawa CSU-X1 spinning disk and an Olympus UPLSAPO 60xs silicone oil immersion objective. GFP and mCherry channels were acquired simultaneously on a pair of aligned EMCCD cameras (C9100-13). Image acquisition was performed using MetaMorph software (Molecular Devices). Embryos were imaged every 75 s, with 30 z slices at 1 μm apart. Lineage tracing and quantification of marker expression were done with the StarryNite and AceTree software as described (Du et al., 2015 (link)).
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