1 × 105 cells were plated onto fibronectin coated coverslips in 6-well plates and transfected with various plasmids or siRNA. Plasmids were transfected 16 h prior to analysis, siRNA 48 h prior to analysis. Cells were starved when indicated and fixed with either PFA or −20°C methanol before blocking and probing with primary and secondary antibodies (Alexa 488, 568 and 633 conjugated anti-mouse or anti-rabbit secondary antibodies, Thermoscientific). Widefield fluorescence images were collected using a Zeiss Olympus IX-81 microscope with either a 40× or 100× objective running Metamorph. Confocal images were collected using a Nikon A1 system with a 100× objective running NIS Elements. Super-resolution imaging was performed an N-SIM Super resolution system. The spinning disc system is described below.
A1 system
Manufactured by Nikon
Sourced in Japan
The Nikon A1 system is a high-performance confocal laser scanning microscope designed for advanced imaging applications. It features a modular design that allows for customization to suit specific research needs. The system provides high-resolution, multi-dimensional imaging capabilities, including time-lapse, 3D, and 4D imaging.
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Lab products found in correlation
Ros assay kit, by Beyotime (1 mentions)
C9205, by Merck Group (1 mentions)
Ab9610, by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
8453 diode array spectrophotometer, by Hewlett-Packard (1 mentions)
500 mhz spectrometer, by Agilent Technologies (1 mentions)
300 mhz spectrometer, by Agilent Technologies (1 mentions)
Fluoromax 4 spectrofluorometer, by Horiba (1 mentions)
Ix81 microscope, by Zeiss (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Anti myc, by Merck Group (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Bz 9000, by Keyence (1 mentions)
Tu plan fluor100, by Nikon (1 mentions)
Lu n4 laser unit, by Nikon (1 mentions)
Eclipse ni, by Nikon (1 mentions)
Alexa flour 594, by Thermo Fisher Scientific (1 mentions)
Anti human cxcr4 antibody, by Abcam (1 mentions)
Anti ki67 antibody, by Agilent Technologies (1 mentions)
11 protocols using a1 system
1
Immunofluorescence Imaging of Transfected Cells
2
Intracellular ROS Detection by DCFH-DA
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Intracellular ROS level was determined with ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were equipped with 10 mM DCFH-DA 30 min before radiation. Upon entry into cells, DCFH-DA is hydrolyzed by cellular esterase into DCFH, which then transfers to fluorescent DCF in response to radiation-induced ROS. Immunofluorescence images were taken using a confocal laser scanning microscope (CLSM, Nikon-A1 system, Dongjing, Japan).
3
Quantifying Macrophages in Tumor Samples
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Perfusion fixed py8119-lentiGFP tumor samples from mice that were treated with Cy5:Cy7 RACPP were suspended in 20% sucrose solution overnight at 4C prior to embedding in OCT solution. 10μm sections were made and treated with a 1:1000 dilution of Alexa405 conjugated primary antibody to F4/80 marker for macrophages (Abcam, Cambridge, UK). The slides were placed in a humidifier chamber overnight at 4C followed by washes with PBS and coverslipped. Three color confocal imaging was performed using the Nikon A1 system with laser lines 405nm, 488nm and 640nm.
4
Optic Nerve Histopathology Analysis
5
Spectroscopic Characterization of Synthesized Compounds
6
Endosome Localization of SKIP Protein
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HeLa cells were plated at a density of 1×105 per well on fibronectin-coated glass coverslips in a six-well plate 6 h prior to transfection. Cells were transfected with 0.8 μg SKIP DNA (CB6 expression vectors) using Effectene transfection reagent (Qiagen). The cells were then incubated at 37°C for 16 h. Cells were fixed at −20°C with 100% methanol for 10 min. This was followed by blocking with 1% BSA in PBS for 20 min. Cells were washed three times with PBS. Cells were then probed with the appropriate antibody (LAMP1, Cell Signaling, D2D11, 1:200 or anti-Myc, Sigma, 9E10, 1:400) diluted in blocking solution (1% BSA in PBS). After 2 h at room temperature, cells were washed three times for 5 min each time with PBS followed by incubation with a secondary fluorescent-protein-conjugated antibody (in blocking solution) for 30 min. Coverslips were washed three more times with PBS, placed cell side down in Fluor save reagent (Calbiochem). Widefield fluorescence images were collected using a Zeiss Olympus IX-81 microscope with a 40× objective running Metamorph software. Confocal images were collected using a Nikon A1 system with a 100× objective running NIS Elements.
7
Fluorescence Imaging of DNAT-Me Uptake
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M7609 cells cultured in chamber slides were incubated with 10 μM DNAT-Me (0.5% DMSO in PBS) and 1 μM SYTO60 for nuclear counterstaining without fixation for 15 min. They were washed with PBS twice, and observed by confocal microscopy (A1 system, Nikon, Tokyo, Japan) with excitation at 488 and 652 nm, respectively. Fluorescence emission spectra of cells incubated with DNAT-Me were measured spectrophotometrically from 470 to 600 nm with excitation at 488 nm. A recombinant GSTP1-1 and glutathione were added to a DNAT-Me (200 μM) solution in a quartz cuvette, and fluorescence emission spectra were measured from 470 to 600 nm with excitation at 488 nm.
For the evaluation of fluorescence intensities, 1 × 105 cells seeded on 35-mm dishes were incubated with DNAT-Me (10 μM) for 10 min, and observed by fluorescence microscopy with excitation at 488 nm (BZ 9000, Keyence Corp. Osaka, Japan). The fluorescence intensity of individual cells in each cell line was quantitated using software provided by the manufacturer. At least 150 cells were quantitated for each cell line and each experiment was repeated three times.
For the evaluation of fluorescence intensities, 1 × 105 cells seeded on 35-mm dishes were incubated with DNAT-Me (10 μM) for 10 min, and observed by fluorescence microscopy with excitation at 488 nm (BZ 9000, Keyence Corp. Osaka, Japan). The fluorescence intensity of individual cells in each cell line was quantitated using software provided by the manufacturer. At least 150 cells were quantitated for each cell line and each experiment was repeated three times.
8
Confocal Laser Scanning Fluorescence Imaging of Liquid Crystal Elastomers
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A confocal laser scanning fluorescence microscope (A1+ system, Nikon) was used to obtain confocal PFOM images of polydomain sample at 25 °C (nematic) with the higher 3D optical resolutions. An optically pumped semiconductor laser (LU-N4 Laser Unit, Nikon, equipped with Sapphire 488, Coherent Inc.) was used to excite the fluorescent probes at 488 nm and the emitted light between 525 and 595 nm was collected. An objective lens with an NA of 0.90 (TU PlanFluor100 ×, Nikon) was used. The confocal PFOM images were acquired at the voxel size of typically 30(x) × 30(y) × 50(z) nm3. The excitation laser was linearly polarised and emitted fluorescence in the same polarisation was collected using a polariser. The obtained images were properly deconvoluted using the associated software. In the present LCEs with dyes, image slices from the top surface to a few µm depth were properly taken with sufficient fluorescent intensity.
9
Aortic Valve Histology and Immunostaining
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Aortic valves removed during surgery were immediately fixed in 10% neutral buffered formalin for 24 h and then embedded in paraffin. For histology and immunostaining, 5-µm sections were cut using a microtome (SLEE medial, Mainz, Germany). Movat pentachrome stain (modified Russell-Movat) (Abcam, Cambridge; UK) was performed according to manufacturer’s instructions. For immunofluorescence analysis, after antigen retrieval slices were incubated overnight with an anti-COX-2 and anti-α-SMA antibodies and then washed and incubated with Alexa fluor-conjugated secondary antibodies. Immunofluorescence images were taken with a confocal microscope (Nikon A1 system) using 20X objective. Pentachrome staining images were taken with a microscope (Nikon Eclipse Ni) using a 10X objective. Procedures are described in detail in Supplementary Methods .
10
Immunohistochemical Detection of Fibrocytes
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The lungs were fixed in 10% buffered formalin and embedded in paraffin. Sections (3 to 4 μm) were stained with hematoxylin and eosin. Paraffin-embedded lung sections were stained to detect fibrocytes using the following antibodies : biotin-conjugated anti-CD45 antibody (Cell Signaling Tech, Danvers, MA, USA), secondary antibody : streptavidin-FITC (eBioscience, Ireland, UK) ; anti-collagen-type 1 antibody (Abcam ; Cambridge, UK) and anti-FSP-1 antibody (Thermo Fisher scientific Inc.), and secondary antibodies conjugated with Alexa Flour 594 or 647 (Thermo Fisher scientific Inc.), respectively.
For triple staining to detect PDGF-BB in fibrocytes, the following antibodies were used : anti-human FSP-1 antibody, (Thermo Fisher scientific Inc.), anti-human CXCR4 antibody (Abcam) and anti-human PDGF-BB antibody (Novus biological, Litteleton, CO, USA), and the fluorescent secondary antibodies conjugated with Alexa Flour 488, 594 or 647 (Thermo Fisher scientific Inc.), respectively. To detect proliferating cells, the section was first stained with anti-Ki-67 antibody (Agilent Technologies) followed by the secondary antibody conjugated with Alexa Flour 594 were used. Images were taken using a confocal laser scanning microscope, A1 system (Nikon, Tokyo, Japan).
For triple staining to detect PDGF-BB in fibrocytes, the following antibodies were used : anti-human FSP-1 antibody, (Thermo Fisher scientific Inc.), anti-human CXCR4 antibody (Abcam) and anti-human PDGF-BB antibody (Novus biological, Litteleton, CO, USA), and the fluorescent secondary antibodies conjugated with Alexa Flour 488, 594 or 647 (Thermo Fisher scientific Inc.), respectively. To detect proliferating cells, the section was first stained with anti-Ki-67 antibody (Agilent Technologies) followed by the secondary antibody conjugated with Alexa Flour 594 were used. Images were taken using a confocal laser scanning microscope, A1 system (Nikon, Tokyo, Japan).
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