For acquiring, processing, and analyzing SIM images on 7W cells and mouse brain tissue, a similar protocol to Hong et al. (2015) was followed. In brief, all samples were imaged using a microscope (ELYRA PS1; Carl Zeiss) with a fixed number of grating rotations (five), and images were processed using the accompanying Zen image acquisition software (Carl Zeiss). For quantification of mouse hippocampal images, the processed 3D SIM images were analyzed by Imaris (bitplane) and MATLAB (MathWorks Inc.) using the spot (ellipsoids) function. Spots were created at the local maxima of all fluorescent puncta spots, and x, y, and z diameters of the ellipsoids were empirically determined for the particular set of antibodies used and confirmed using spot growth boundary so that all spots created appropriately reflected the fluorescent image of each channel. Then, MATLAB was used to determine the number of colocalized spots (≤200-nm distance between spot centers of two channels). The number of colocated spots was then divided by the numbers of spots on the individual channel. All SIM data analysis was done blinded.
Zen image acquisition software
Manufactured by Zeiss
Sourced in Germany
The Zen image acquisition software is a core component of Zeiss microscopy systems. It provides a user-friendly interface for controlling the microscope hardware and capturing high-quality images. The software's primary function is to facilitate the acquisition and storage of digital images from various Zeiss microscope models.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m2m, by Zeiss (2 mentions)
Axiocam 503 mono, by Zeiss (2 mentions)
Matlab, by MathWorks (1 mentions)
Elyra ps 1, by Zeiss (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope 700, by Zeiss (1 mentions)
Alexa 488 conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Axioskop2 lsm510, by Zeiss (1 mentions)
880 airyscan confocal microscope, by Zeiss (1 mentions)
Superfrost plus adhesion slides, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ab205921, by Abcam (1 mentions)
35 mm glass bottomed dishes, by MatTek (1 mentions)
8 protocols using zen image acquisition software
1
Super-Resolution Imaging of Synaptic Proteins
2
Bacterial Infection Imaging Protocols
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For micrographs of bacteria on beads, GFP-LVS and/or DsRed labeled LVS were coated onto the beads as described above. The bacteria bound beads were fixed in 4% paraformaldehyde and were placed into an 8 well chamber slide (Nunc). The beads were allowed to settle and the fixative was carefully removed.
For images of infected cells, beads were prepared as above with the indicated bacteria. J774 cells were inoculated for 2 hours and then the media was removed and replaced with media containing 25 µg/ml of gentamicin. The cells were washed and fixed with 4% paraformaldehyde at 4 or 22 hours post inoculation. Cells were treated with 50 mM ammonium chloride for 10 minutes and then stained with 10 µg/ml of AF647 wheat germ agglutinin (Invitrogen) where indicated.
All samples were mounted using a DAPI containing mounting media (Vector Shield). Images were acquired using a Zeiss 700 confocal laser scanning microscope (Zeiss) using Zen image acquisition software (Zeiss) or an Olympus FV500 confocal scanning laser microscope (Olympus). Images were cropped and scale bars were added using ImageJ [28] .
For images of infected cells, beads were prepared as above with the indicated bacteria. J774 cells were inoculated for 2 hours and then the media was removed and replaced with media containing 25 µg/ml of gentamicin. The cells were washed and fixed with 4% paraformaldehyde at 4 or 22 hours post inoculation. Cells were treated with 50 mM ammonium chloride for 10 minutes and then stained with 10 µg/ml of AF647 wheat germ agglutinin (Invitrogen) where indicated.
All samples were mounted using a DAPI containing mounting media (Vector Shield). Images were acquired using a Zeiss 700 confocal laser scanning microscope (Zeiss) using Zen image acquisition software (Zeiss) or an Olympus FV500 confocal scanning laser microscope (Olympus). Images were cropped and scale bars were added using ImageJ [28] .
3
Immunofluorescence Analysis of Protein Localization
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Cells were grown on glass coverslips in 35 mm plates for 24 h. After incubation with TAT-fused proteins (3 μM) in serum-free DMEM-F12, cells were thoroughly washed 4 times with PBS and fixed with 4% PFA-Sucrose in PBS for 30 min. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS and subsequently blocked with 0.5% BSA in PBS for 1 h. Cells were then incubated with rabbit anti-LepFNR (1:50) and anti-LepHO (1:200) antibodies for 1 h at room temperature. The coverslips were washed, incubated 1 h at room temperature with the secondary Alexa-488 conjugated anti-rabbit antibody (1:800, Life Technologies Corporation, Carlsbad, CA, USA) and mounted with ProLong® with DAPI. A sample was processed in parallel omitting the primary antibody as a background control. Confocal images were obtained using a Zeiss LSM 880 confocal microscope with a 20X objective. Zen image acquisition software (Carl Zeiss) was used to analyze images.
4
Imaging Apical Membrane Dynamics
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Transfected W4 cells were split and seeded onto glass-bottom dishes (WillCo Wells) in the presence of 1 µg/ml doxycycline. Before imaging, medium was removed and replaced with Hepes-buffered (pH = 7.4) Leibovitz’s L-15 medium (Invitrogen). Cells were imaged at 37°C using an Axioskop2 LSM510 scanning confocal microscope (Carl Zeiss) with a 63× magnification oil objective (Plan Apochromat, NA 1.4) using Zen image acquisition software (Carl Zeiss). Apical membrane size was determined using ImageJ software (National Institutes of Health) by measuring the total perimeter of the cell and the length of the membrane bearing microvilli. Mean apical membrane sizes were compared using an independent samples t test in SPSS with a p-value <0.05 as a cutoff for significance. Quantification of apical enrichment of YFP-Cdc42 was performed by making a line scan through the apical and basal membrane and determining the ratio between mean apical and basal membrane pixel intensities using ImageJ. Mean enrichment ratios were compared using an independent samples t test in SPSS with a p-value <0.05 as a cutoff for significance.
5
Automated Enrichment and Staining of Circulating Tumor Cells
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CTCs were enriched and immunostained using an automated MCA system.18 , 25 , 26 A whole blood sample was added to the reservoir of the MCA system. The blood sample was filtrated through the metal filter in the cartridge. The staining process is automated as follows: CD45 staining for 1 h, fixation for 10 min, permeabilization for 10 min, AXL staining for 90 min, and finally DAPI and CK or VM staining for 30 min. Four‐minute washes were carried out between each step and a 7‐min wash was done before the final step. Cell fixation, permeabilization, wash buffer and staining reagents for CD45, DAPI, and CK were provided by Hitachi chemical company. AXL was stained with the same antibody as immunocytochemistry and VM was stained with vimentin V9 Alexa Fluor 488 (sc‐6260 AF488, Santa Cruz Biotechnology). An image of the entire cell array area was captured using a fluorescence microscope (Axio Imager M2m; Carl Zeiss) with an integrated 10× objective lens and a computer‐operated motorized stage, a digital camera (AxioCam 503 mono; Carl Zeiss), and ZEN image acquisition software (Carl Zeiss). CTCs were defined as DAPI, CK or VM‐positive, and CD45‐negative cells.
6
Quantifying Neurogenesis with Immunofluorescence
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For quantification of neurogenesis, 7 brain sections with intersection distance 240 µm were co-labeled with immunofluorescence markers of cellular proliferation (BrdU) and mature neurons (hexaribonucleotide binding protein-3 (NeuN)). Sections were incubated with primary antibodies (BrdU mouse monoclonal antibody and NeuN rabbit monoclonal antibody) at 4°C overnight. The following day, the sections were incubated for 60 min in a solution of Tris-buffered saline containing fluorescently conjugated secondary antibodies (Goat anti-Mouse IgG H&L Alexa Fluor 488 and Goat anti-Rabbit IgG H&L Alexa Fluor 594) and mounted on SuperFrost Plus Adhesion Slides (Thermo Fisher Scientific).
For all sections, Z-stack images of the dorsal dentate gyrus (bregma range –3.14 to –4.5229 ) were bilaterally imaged with Zeiss 880 Airyscan Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). The Zen image-acquisition software (Carl Zeiss AG) enabled a reusable imaging routine setup with the experiment designer module. The image analyses for quantification of neurons and neurogenesis were done using Fiji software.30 (link) For more details, seeSupplementary Materials .
For all sections, Z-stack images of the dorsal dentate gyrus (bregma range –3.14 to –4.5229 ) were bilaterally imaged with Zeiss 880 Airyscan Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). The Zen image-acquisition software (Carl Zeiss AG) enabled a reusable imaging routine setup with the experiment designer module. The image analyses for quantification of neurons and neurogenesis were done using Fiji software.30 (link) For more details, see
7
Automated Enumeration and Characterization of CTCs
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CTCs were enriched and immunostained using an automated MCA system [30 (link),31 (link),32 (link)]. Peripheral whole blood sample was added to the reservoir of the MCA system, and then the blood sample was filtered through the metal filter in the cartridge. After tumor cells were captured, the cells were automatically stained for CD45, PD-L1, CK, and 4′,6-diamidino-2-phenylindole (DAPI). The reagents for CD45, CK, and DAPI staining were provided by Hitachi Chemical Company (Chikusei, Japan). PD-L1 was stained with 1:1000 diluted anti-PD-L1 rabbit mAb (28–8) (ab205921; Abcam, Cambridge, MA, USA) and then incubated with 1:500 diluted goat-anti-rabbit Alexa Fluor 647 (A-21245; Thermo Fisher Scientific, Waltham, MA, USA). An image of the entire cell array area was captured using a fluorescence microscope (Axio Imager M2m; Carl Zeiss, Oberkochen, Germany) with an integrated 10× objective lens and a computer-operated motorized stage, a digital camera (AxioCam 503 mono; Carl Zeiss), and ZEN image acquisition software (Carl Zeiss). CTCs were defined as DAPI, CK-positive, and CD45-negative cells. PD-L1-positive or -negative CTCs were counted by two people independently for each acquired image. The proportion of PD-L1-positive CTCs in the detected total CTCs were defined as PD-L1 positivity rate for each sample.
8
Folding Pulse Experiments: Monitoring EPAC FRET
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For folding pulse experiments, cells were placed in glass-bottomed 35-mm dishes (MatTek) and transfected with the VSVG-mcherry plasmid and the EPAC YFP/CFP FRET-pair for 16 h at 40 C, or the cells were co-microinjected and then kept for 3 h at 40 C in DMEM-HEPES. Before the experiment, the cells were incubated at 40 C with cycloheximide (100 mg/ml) for 30 min and then mounted on a Zeiss LSM710 laser scanning microscope at 40 C, using a water-jacketed stage to regulate the temperature and running the Zen image acquisition software (Zeiss). The temperature was kept at 40 C for 10 min before the start of the traffic pulse. Basal cAMP and PKA activities were recorded over 3-5 min at 40 C, and then for 10 min of traffic pulses at 10-30 s interval, using a 458-nm laser for excitation (CFP), and with simultaneous acquisition of CFP (480 nm) and YFP (514 nm) emissions (sensitized FRET). Sequential acquisition of VSVG-mcherry was carried out using 568-nm-wavelength lasers for excitation, with the 63 3 oil-immersion objective. The CFP:YFP ratio was then quantitated (see below).
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