Day 6 myotubes expressing Halo‐Myh3 were labeled with Oregon‐Green®︎ ligand (green fluorescence; Promega) at a final concentration of 0.3% in DM for 16 h. Following washout, myotubes were reacted with TMR ligand (red fluorescence, Promega) at a final concentration of 0.1% in minimum essential medium (Thermo Fisher Scientific) to label newly synthesized Halo‐Myh3 for 7, 15, and 30 min. Myotubes were fixed with 4% paraformaldehyde in PBS (Nacalai Tesque, Kyoto, Japan), rinsed with 0.5% Triton X‐100 in PBS for 5 min three times, and mounted with media containing 4’,6‐diamidino‐2‐phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Samples were analyzed using an LSM 700 Confocal Laser Scanning Microscope (Carl Zeiss, Tokyo, Japan) equipped with a Plan‐Apochromat ×63 (numerical aperture 1.4) lens. The DAPI, Oregon‐Green, and TMR fluorescence were detected at excitation wavelengths of 405, 488, and 555 nm with 300–483, 493–550, and 560–800 nm band‐pass filters, respectively. Images were processed by using zen 2012 imaging software (Carl Zeiss).
Zen 2012 imaging software
Manufactured by Zeiss
Sourced in Germany
ZEN 2012 is an imaging software developed by Zeiss. The software provides core functions for image acquisition, processing, and analysis. It is designed to work with a variety of Zeiss microscopy systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (6 mentions)
Donkey fab fragments, by Jackson ImmunoResearch (3 mentions)
Cleaved caspase 3 cc3, by Cell Signaling Technology (2 mentions)
β galactosidase, by Merck Group (2 mentions)
Cy3 conjugated goat anti rat igg secondary antibody, by Beyotime (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Histo, by Zeiss (2 mentions)
Prism 9, by GraphPad (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Plan apochromat 63, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Steponeplus instrument, by Thermo Fisher Scientific (1 mentions)
19 protocols using zen 2012 imaging software
1
Halo-Myh3 Protein Synthesis Tracking
2
Midgut Dissection and Immunostaining
3
Imaging Fungal Hyphae and Gene Expression
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For imaging of hyphae, cells were grown on coverslips in 0.3 ml AMM containing 1% (w/v) glucose, 20 mM glutamine and the desired iron concentration. Microscopy images were captured using an Axio Imager. M2 microscope (Carl Zeiss) equipped with a 63× oil immersion objective lens (numerical aperture, 1.40), a HPX 120 V compact light source (Carl Zeiss) and the AxioCam MRm camera (Carl Zeiss). Images were processed using ZEN 2012 imaging software (Carl Zeiss). DAPI was used to stain nuclei. PpIX content, siderophore production, RNA isolation, and Northern analysis were carried out as described previously (Hortschansky et al, 2007 (link)). The hybridization probes used in this study were generated by PCR using DIG-labeled nucleotides. For qRT-PCR analysis, RNA was digested with DNase I and column eluted using RNA Clean & Concentrator™-25 kit (ZYMO Research). cDNA was synthesized using GoScript™ Reverse Transcription System (Promega) and random primers. qPCR was performed in a StepONE Plus Instrument (Applied Biosystems) with POWER SYBR® Green PCR Master Mix (Applied Biosystems). Primers for hapX, sreA, and actA are listed in Supplementary Table S4.
4
Immunocytochemistry of PBMCs with rFgRab10 Protein
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PBMCs were incubated with rFgRab10 protein in a humidified atmosphere of 5% CO2 at 37 °C for 1 h. Cells were fixed with 4% paraformaldehyde, washed 3 times in PBS and treated with blocking solution (4% BSA in PBS) for 1 h to minimize background staining. rFgRab10-treated, SUMO protein-treated, or PBS-treated PBMCs were incubated with rat anti-rFgRab10 protein antibody (1:100 in 4% BSA) for 1 h at 37 °C. Cells were stained with Cy3 conjugated goat anti-rat IgG secondary antibody (1:500 in 4% BSA) (Beyotime, Jiangsu, China) for 1 h at 37 °C. Hoechst 33342 (Invitrogen, Oregon, USA) was used to stain the nuclei. Stained cells were imaged using a confocal laser scanning microscope (LSM710, Zeiss, Jena, Germany) and digital images were analyzed using Zen 2012 imaging software.
5
Quantification of Confocal Imaging for Research
6
Midgut Dissection and Immunostaining
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Intact female midguts were dissected using standard protocols59 (link). Primary antibodies were: anti Dronc60 (link) (1:200; a kind gift of Pascal Meier); PH3 (1:2,000; Millipore), Prospero (Pros, 1:20; DSHB; Prospero (MR1A) was deposited to the DSHB by C.Q. Doe); Pdm-161 (link) (1:1,000; a kind gift of Yu Cai). DAPI was used to counterstain nuclei. Phalloidin labeling was used to assess the physical properties of the guts. Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab35 (link),36 (link) in the posterior midgut was imaged and analysed. Images were obtained with a Zeiss LSM 700 confocal microscope, analysed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.
7
Immunofluorescent Detection of rFg14-3-3e in Goat PBMCs
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Freshly isolated goat PBMCs were incubated with rFg14-3-3e protein for 1 h in a humidified atmosphere of 5% CO2 at 37 °C. To minimize background staining, goat PBMCs were fixed with 4% paraformaldehyde, washed 3 times in PBS (5 min each) and were treated with blocking solution (4% BSA in PBS) for 1 h at ambient temperature. rFg14-3-3e-treated or non-treated control goat PBMCs were incubated with rat anti-rFg14-3-3e antibody (1:100) for 1 h at 37 °C, followed by staining with Cy3 conjugated goat anti-rat IgG secondary antibody (1:500) (Beyotime, Haimen, Jiangsu, China) for 1 h at 37°C. Hoechst 33342 (Invitrogen, Eugene, Oregon, USA) was used for nuclear staining. Stained cells were imaged with 100× magnification using a Zeiss laser scanning confocal microscope (LSM710, Zeiss, Jena, Germany) and digital images were analyzed by Zen 2012 imaging software.
8
Midgut Dissection and Immunostaining
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Intact female midguts were dissected using standard protocols (Amcheslavsky et al., 2009 (link)). Primary antibodies were: PH3 (Millipore: 1:2,000), β-Galactosidase (DSHB; 1:100 (concentrate)), NimC (Kurucz et al., 2007 (link); 1:300); Dronc (SK11) (Wilson et al., 2002 (link); 1:200); cleaved caspase 3 (CC3) (Cell Signaling Technology; 1:400). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab in the posterior midgut was imaged and analyzed. Images were obtained with a Zeiss LSM 700 confocal microscope, analyzed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.
9
Visualization and Quantification of DNA Damage and Autophagy
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Cells on glass coverslips were treated with either MMV652103 or vehicle for 24 and 48 h and processed for immunofluorescence with antibodies against phospho H2A.X (#2577) (1:500) or LC3B (#2775) (1:200) (Cell Signaling Technology) as previously described (Bleloch et al., 2019[8 (link)]). Cells were imaged using confocal microscopy (Carl Zeiss LSM 880 with Fast Airyscan module confocal (Oberkochen, Germany)). Multiple z layers were acquired with 1 μM step width. Images were processed using ZEN 2012 imaging software (Carl Zeiss) and maximum intensity projections were generated. For quantification, mean fluorescence was measured from at least 20 fields of view per treatment condition and pooled from three independent replicates.
10
Multimodal Microscopy Imaging of Cells
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Images of H&E and IHC staining were taken by an Axio Lab 1 microscope using 10×, 20×, and 40× Zeiss A-Plan objectives and were captured using a Canon EOS 1000D camera and AxioVision software (Carl Zeiss). Images of IF staining and organoids were acquired on a Nikon ECLIPSE E800 epi-fluorescence microscope at 10×, 20×, and 40× Nikon Plan Fluor objectives using an QImaging RETGA EXi camera with QCapture software (QImaging). Fluorescence images for nuclear pore complex were collected using Zeiss LSM-700 confocal microscope with Zen 2012 Imaging Software.
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