Hela cells grown on glass coverslips in 6-well plates were co-transfected with HA-IFITM3 constructs and LAMP1-mCherry for 16 h, and treated with Bafilomycin A1 or ML240 for 12 h. Cells were then fixed with 3.7% (w/v) paraformaldehyde, permeabilized with 0.1% (w/v) Triton X-100, and blocked with 5% (w/v) BSA in PBS. Cells were incubated with anti-HA antibody conjugated to AlexaFluor-488 (1:800) in 0.05% Triton X-100 in PBS at 4°C overnight and washed with PBS three times. Finally, the coverslips were mounted onto slides using ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific) and examined by an inverted LSM 780 laser scanning confocal microscope (Zeiss) with a Zeiss Plan-Apochromatic 63×/1.4 N.A. oil immersion objective. Images were acquired and analyzed with the ZEN blue 2012 software (Zeiss). Pearson's correlation coefficients were calculated in ZEN blue 2012 software (Zeiss).
Zen blue 2012
Manufactured by Zeiss
Sourced in Germany, United States
ZEN blue 2012 software is a microscope control and image acquisition software developed by Zeiss. It provides a user-friendly interface for controlling Zeiss microscopes and capturing high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (12 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (7 mentions)
Lsm 710, by Zeiss (6 mentions)
Flash 4.0 camera, by Hamamatsu Photonics (3 mentions)
Histoclear, by Merck Group (3 mentions)
Guinea pig anti insulin, by Agilent Technologies (3 mentions)
Mouse anti glucagon, by Merck Group (3 mentions)
Plan apochromat 206 0.8 m27 air objective, by Hamamatsu Photonics (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Axio imager z2, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Axio imager, by Zeiss (2 mentions)
C apochromat 20x objective, by Zeiss (2 mentions)
Apotome 2, by Zeiss (2 mentions)
Microm hm 355, by Thermo Fisher Scientific (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Chamber slide, by Ibidi (1 mentions)
36 protocols using zen blue 2012
1
Visualization of IFITM3 and LAMP1 co-localization
2
IFITM3 Localization in Hela Cells
3
Epididymal Adipose and Liver Tissue Analysis
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Epididymal white adipose tissue (eWAT) and livers were collected. Samples were stored at −80°C until processed. While frozen, eWAT was divided in half. One half of the frozen tissue was fixed in 10% buffered formalin at 4°C for 48 hours.20 The other half was immediately immersed in Ribozol for RNA extraction (see below RNA section). Once fixed, a total of four randomly selected samples per group were paraffin‐embedded and sliced at 5 µm using a microtome (Microm HM 355S; Thermo Scientific). Three consecutive sections per paraffin block were cut and mounted per slide. A total of three sections per animal were processed and analysed (n = 4 animals/group). Standard haematoxylin and eosin staining was performed. Tissue was scanned using the Carl Zeiss Axioscan Z1 (10×/0.45, M27 Plan Apochromat objective lens) and the Carl Zeiss Zen Blue (2012) software.
4
Evaluating Antioxidant Effects of L. viridis Essential Oil
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RAW 264.7 cells (5 × 104 cells/well) were plated in a µ-Chamber slide (IBIDI GmbH, Germany), allowed to stabilize overnight and then treated with LPS (1 μg/mL) during 16 h. When indicated, L. viridis L´Hér. essential oil (0.64 μL/mL) was added 1 h prior to LPS. At the end of the incubation period, cells were washed three times and then loaded with 5 μM H2DCFDA (fluorescent indicator of ROS) and 0.5 μg/mL Hoechst (DNA stain) in HBSS for 30 min at 37°C in the dark. Cells were washed with HBSS and analysed using an Zeiss Axio Observer.Z1 inverted microscope (Carl Zeiss, Germany) equipped with an AxioCam HRm and Zen Blue 2012 software, using a 63× oil objective (Plan-Apochromat, 1.4 NA).
5
Immunofluorescence Analysis of Mouse Skin Samples
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Punch biopsies were taken from harvested mouse skin and frozen at –20°C. Sections of 8 μm were generated on glass microscope slides (Star Frost, Laborchemie GmbH, Vienna, Austria) with a cryotome (MICROM HM 550, Thermo Fisher Scientific Inc., Runcorn, UK). For histological analysis sections were stained with hematoxylin (Merck) and eosin (Merck). For immunofluorescence staining sections were fixed in ice cold aceton (Merck) for 2 min and blocked in 0.5% BSA (Sigma-Aldrich) in PBS (Invitrogen) for 60 min. Primary antibodies were: pAb against the NC-1 domain of type VII collagen (LH7.2, kindly provided by Dr. Alexander Nyström from Freiburg) produced in rabbit diluted 1:3000 in PBS for 90 min, monoclonal anti-involucrin (Santa Cruz Biotechnology, Heidelberg, Germany), 1:100 for 2 h. The secondary antibody anti-rabbit AF594 and anti-mouse AF488 (Invitrogen) were incubated for 1 h in a 1:400 dilution in PBS in the dark. DAPI (Sigma-Aldrich) staining of the nuclei (Sigma-Aldrich) was performed in dilution of 1:2000 for 10 min. After mounting, using a fluorescence aqueous mounting medium (Dako), the sections were observed under the confocal laser scanning unit (Axio Observer Z1 attached to LSM710, Zeiss) and images were converted to TIFF files with the ZEN Blue 2012 (Carl Zeiss) software.
6
Microscopy Imaging Protocol with Zeiss
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Imaging was performed in a Zeiss Observer Z.1 inverted microscope equipped with 5× (N-Achroplan, 0.15 M27), 20× (Plan-Apochromat, 0.8 NA) and 40× (LD Plan-Neofluar, 0.6 Corr Ph2 M27) objectives, coupled to a AxioCam HRm camera and Zen Blue 2012 software (all from ZEISS, Oberkochen, Germany). All conditions within an experiment were processed simultaneously and imaging settings (exposure time to LED excitation) were conserved. Images were analyzed using the public domain software Fiji (ImageJ) (https://fiji.sc ).
7
Microscopic Examination of Cochlear Tissue
8
Time-Lapse Imaging of GFP Chicken Explants
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For time-lapse imaging, E6.5 explants prepared from CAG-GFP chicken embryos were mounted onto nitrocellulose filters and cultured skin side down in a 6-well dish containing 2 ml standard or supplemented medium at 37°C, 5% CO2. Real-time imaging was performed using the Zeiss Live Cell Observer/Deconvolution system, imaged every 10 minutes for the duration of the experiment, and analysed using ZenBlue2012 (Zeiss) software.
For the PIV analyses [77 ,78 (link)], microscope image frames of skin showing fluorescent (red and green) cells were superposed in one image to achieve higher density. PIV was conducted using MatPIV, implemented in Matlab, with global, local, and signal-to-noise filter. Interrogation areas (32 × 32 pixel size) with 50% overlap were chosen. PIV yields displacement (velocity) fields for every pair of consecutive frames, which were used to derive velocity and angle kymographs as well as streamlines.
For the PIV analyses [77 ,78 (link)], microscope image frames of skin showing fluorescent (red and green) cells were superposed in one image to achieve higher density. PIV was conducted using MatPIV, implemented in Matlab, with global, local, and signal-to-noise filter. Interrogation areas (32 × 32 pixel size) with 50% overlap were chosen. PIV yields displacement (velocity) fields for every pair of consecutive frames, which were used to derive velocity and angle kymographs as well as streamlines.
9
Quantifying Fluorescence in Electrode Traces
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We measured the fluorescence of the electrode traces seen in the confocal images (Fig. 3 ) on unadjusted MIPs. The mean intensity in 80 μm × 600 μm rectangles placed on the identifiable electrode traces were subtracted the mean background intensity in a 160 μm × 600 μm rectangle placed elsewhere on the tissue in the images. This was done in Zen Blue 2012 (Zeiss). Measurements were handled in Prism 6 (GraphPad).
10
Residual Tumor Burden Measurement in Prostate Cancer
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Residual tumor was concordantly identified by three board-certified genitourinary pathologists (M.M., R.L., and H.Y.). Residual cancer burden (RCB)30 (link) was measured and calculated by multiplying the number of slices through which each residual tumor extended by the largest cross-sectional width and length and block thickness (0.6 cm). Volume was further corrected by multiplying by 0.4 and the estimated tumor cellularity. Precise length and width measurements were performed on scanned slides using Zen Blue 2012 (Zeiss) with objective/magnification and pixel:distance calibrations recorded within the scanned CZI file. The right-sided lesion extended through four blocks. With a length of 2.09 cm, a width of 1.73 cm, and 10% cellularity, the RCB was 0.35 cc. The left-sided lesion extended through two blocks. With a length of 0.62 cm, a width of 0.35 cm, and 10% cellularity, the RCB was 0.01 cc.
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