Cells were fixed with a solution of 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich; St. Louis, MO) and treated to prevent nonspecific binding (BlockAid, Life Technologies; Carlsbad, CA). Cytoskeletal F-actin was detected with Alexa Fluor 488 conjugated to phalloidin, then mounted with ProLong® Gold Antifade Mountant with DAPI (Life Technologies; Carlsbad, CA) to stain nuclei. Samples cured overnight before imaging. Slides were imaged with a Zeiss LSM 510 Meta system combined with the Zeiss Axiovert Observer Z2 inverted microscope and ZEN 2009 imaging software (Carl Zeiss, Inc., Thornwood, NY). Images were acquired in a single plane utilizing the Plan-Apochromat 20x/NA 0.8 and Fluar 40x/NA 1.30 Oil objectives. Transmitted light was collected on one channel during the z-stack acquisition to provide contrast to the GF structure. Confocal z-stack images were acquired utilizing the Plan-Apochromat 63X/NA 1.4 and alpha Plan-Fluar 100X/NA1.45 Oil objectives. All images were collected with a diode (405 nm) and Argon (488 nm) laser sources and the following band-pass emission filters: BP 420-480 BP 505-530. Images were processed with ZEN 2009 imaging software (Carl Zeiss, Inc., Thornwood, NY).
Zen 2009 imaging software
Manufactured by Zeiss
Sourced in Germany
ZEN 2009 is an imaging software developed by Zeiss. It provides a user interface for controlling and managing microscope systems and acquiring, processing, and analyzing microscopic images.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 laser scanning microscope, by Zeiss (19 mentions)
Rpmi 1640 defined medium, by Merck Group (18 mentions)
Syto17 dye, by Thermo Fisher Scientific (18 mentions)
Lsm 510 meta system, by Zeiss (9 mentions)
Axiovert observer z1 inverted microscope, by Zeiss (5 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lsm 880 inverted confocal microscope, by Zeiss (3 mentions)
Vectashield hardset mounting medium with dapi, by Vector Laboratories (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Blockaid, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Axiovert observer z2 inverted microscope, by Zeiss (2 mentions)
Doxycycline hyclate, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Axiovert observer z1 inverted, by Zeiss (2 mentions)
Lsm 700 laser scanning, by Zeiss (2 mentions)
Lsm 510 duo, by Zeiss (1 mentions)
Plan apochromat 40 1.4 oil dic objective, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
39 protocols using zen 2009 imaging software
1
Immunofluorescence Staining of Cytoskeleton
2
Confocal Imaging of Tau Pathology
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For confocal immunofluorescence imaging, the primary antibodies were visualized with secondary antibodies tagged with either Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen, Carlsbad, CA). Images were taken with a Zeiss LSM 510 Meta system combined with the Zeiss Axiovert Observer Z1 inverted microscope and ZEN 2009 imaging software (Carl Zeiss, Inc., Thornwood, NY). Confocal Z-stack and single plane images were acquired with an Argon (488 nm) and a HeNe (543 nm) laser source. Z-stacks images were acquired using a 20x Plan-Apochromat (NA 0.8) objective, emission band passes of 505–550 nm for the detection of the TauC3 (green channel, Alexa Fluor 488) and 550–600 nm for detection of PHF-1 (red channel, Alexa Fluor 555). All images displayed are 2-D, maximal intensity projections generated acquired Z-stacks. Single plane images were acquired with a 63x Plan-Apochromat oil-immersion objective (NA 1.4) with emission long pass of 505 nm for the detection of the TauC3 antibody (green channel, Alexa Fluor 488) and 550–600 nm for the detection of PHF-1 (red channel, Alexa Fluor 555).
3
Confocal Imaging of nApoECF and PHF-1 in Neurons
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Confocal immunofluorescence imaging was as previously described [23 (link)], and primary antibodies were visualized with secondary antibodies tagged with either Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen, Carlsbad, CA). Images were taken with a Zeiss LSM 510 Meta system combined with the Zeiss Axiovert Observer Z1 inverted microscope and ZEN 2009 imaging software (Carl Zeiss, Inc., Thornwood, NY). Confocal Z-stack and single plane images were acquired with an Argon (488 nm) and a HeNe (543 nm) laser source. Z-stacks images were acquired using a 20x Plan-Apochromat (NA 0.8) objective, emission band passes of 505-550 nm for the detection of the nApoECF (green channel, Alexa Fluor 488) and 550-600 nm for detection of PHF-1 (red channel, Alexa Fluor 555). All images displayed are 2-D, maximal intensity projections generated acquired Z-stacks. The optical depth used varied between experiments but was in the range of 10-25 µm in the Z-plane. Single plane images were acquired with a 63x Plan-Apochromat oil-immersion objective (NA 1.4) with emission long pass of 505 nm for the detection of the nApoECF antibody (green channel, Alexa Fluor 488) and 550-600 nm for the detection of PHF-1 (red channel, Alexa Fluor 555).
4
Inhibition of Pseudomonas aeruginosa Biofilm by Fe-Tart
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Example 4
Materials and Methods
Pseudomonas aeruginosa PAO-1 strain was routinely grown on either LB (Luria-Bertani, Oxoid, UK) agar plates at 37° C. or in broth at 37° C. with 200 rpm shaking. UV-sterilized glass slides were incubated in either 15 mL RPMI-1640 defined medium (Sigma, UK) or 15 mL RPMI-1640 with Fe-Tart inoculated with diluted (OD600=0.01) bacteria from overnight cultures at 37° C. with 60 rpm shaking for 72 hours. The slides were removed from bacterial culture and washed with 15 mL phosphate buffered saline at room temperature for 5 minutes three times and then rinsed with distilled H2O. After washing, the slides were stained with 20 μM SYTO17 dye (Invitrogen, UK) at room temperature for 30 minutes. After removing excess staining dye and air-drying, the samples were examined using a Carl Zeiss LSM 700 Laser Scanning Microscope with ZEN 2009 imaging software (Carl Zeiss, Germany). The coverage rate of bacteria on the surface was analysed using open source Image J 1.44 software (National Institute of Health, US).
Results
5
Fluorescent Microscopy of Basidioascus Meiosis
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To study nuclear behaviour and to look for indicators of meiosis, Basidioascus undulatus DAOM 241956 was grown on corn meal agar (CMA, Acumedia Manufacturers, Lansing, MI) for 1 wk and mounted in DNA stains: DAPI-Fluoromount-GTM mounting medium (EMS, Hatfield, PA) or aqueous SYTO 9 (25 μM) (Life Technologies, Burlington, ON). Samples were visualized under confocal laser scanning microscopy using an LSM 510 DUO (Carl Zeiss MicroImaging, Göttingen, Germany) with a Plan-Apochromat 40×/1.4 Oil DIC objective and electronic zoom 4. An excitation diode laser (405 nm) and emission light (420–700 nm) were used for DAPI. An excitation Argon laser (488 nm) and emission light (505–550 nm) were used for SYTO 9. Images were captured using ZEN 2009 Imaging Software (Carl Zeiss MicroImaging).
6
Immunocytochemistry Protocol for TREx-BCBL-1-RTA Cells
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TREx‐BCBL‐1‐RTA cells were seeded onto poly‐L‐lysine‐coated coverslips and 2 µg/ml doxycycline hyclate (Sigma‐Aldrich) was added 3 h later. Cells were fixed with 15 min with 4% paraformaldehyde and permeabilised with PBS + 1% Triton. All further incubation steps occurred at 37°C. Coverslips were blocked for 1 h with PBS and 1% BSA before 1 h incubation with the appropriate primary antibody and followed by 1 h with Alexa‐Fluor conjugated secondary antibody 488 (Invitrogen 1/500). Coverslips were mounted using Vectashield Hardset Mounting Medium with DAPI (Vector laboratories). Images were obtained using a Zeiss LSM880 Inverted Confocal Microscope and processed using ZEN 2009 imaging software (Carl Zeiss) as previously described (Baquero‐Perez & Whitehouse, 2015 (link)).
7
Confocal Imaging of nZnO Dissolution
8
Quantifying Motor Neuron GFP Expression
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Cells in each quadrant of the ventral horn were counted on z-series of slice images using a confocal microscope and ZEN2009 imaging software (Zeiss). At least 3 embryos were quantified from each group, and 3–4 images were collected from each spinal cords. %GFP/column was calculated as the percentage of the number of GFP-expressing cells among the motor neurons in each column. To quantify GFP intensity induced by exogenous transcription factors in the chick spinal cords after electroporation, 12 μm-thick transverse sections were immunolabeled with GFP. The background-subtracted pixel intensities of GFP in 120 x 240 μm2 areas in the dorsal spinal cord were measured using ImageJ. At least 10 sections from 4 embryos were analyzed for each group. Statistical significance was analyzed by unpaired Student’s t-test and the Kruskal-Wallis test for multiple comparisons.
9
Effect of Iron Citrate on Pseudomonas Biofilm Formation
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Example 3
Materials and Methods
Pseudomonas aeruginosa PAO-1 strain was routinely grown on either LB (Luria-Bertani, Oxoid, UK) agar plates at 37° C. or in broth at 37° C. with 200 rpm shaking. UV-sterilized glass slides were incubated in either 15 mL RPMI-1640 defined medium (Sigma, UK) or 15 mL RPMI-1640 with Fe-Cit inoculated with diluted (OD600=0.01) bacteria from overnight cultures at 37° C. with 60 rpm shaking for 72 hours. The slides were removed from bacterial culture and washed with 15 mL phosphate buffered saline at room temperature for 5 minutes three times and then rinsed with distilled H2O. After washing, the slides were stained with 20 μM SYTO17 dye (Invitrogen, UK) at room temperature for 30 minutes. After removing excess staining dye and air-drying, the samples were examined using a Carl Zeiss LSM 700 Laser Scanning Microscope with ZEN 2009 imaging software (Carl Zeiss, Germany). The coverage rate of bacteria on the surface was analysed using open source Image J 1.44 software (National Institute of Health, US).
Results
10
Immunofluorescence Staining of TREx-BCBL1-Rta Cells
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TREx-BCBL1-Rta cells were seeded onto coverslips treated with poly-l -lysine (Sigma, St. Louis, MO, USA), fixed for 15 min in 4% paraformaldehyde and permeabilised with PBS + 1% Triton-X-100, as previously described [47 (link)]. Subsequent incubations were carried out at 37 °C for 1 h in a humidified chamber. Cells were blocked using PBS with 1% BSA prior to incubation with the desired primary antibodies and then Alexa Fluor-conjugated secondary antibodies. (Invitrogen 1:500, Paisley, UK). Coverslips were mounted onto slides using Vectashield Hardset Mounting Medium with DAPI (Vector laboratories, Newark, NJ, USA). Images were acquired using a Zeiss LSM880 inverted confocal microscope and analysed using ZEN 2009 imaging software (Carl Zeiss, Jena, Germany).
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