Imaging for AgNO3 uptake was carried out with a Zeiss ApoTome.2 microscope using a 20× Plan-Apochromat 0.8 NA air objective. Confocal imaging for Dextran uptake, ANF-RFP, LysoTracker assays and anti-PDI was carried out using a Zeiss LSM900 microscope with a 20× Plan-Apochromat 0.8 NA air objective. Immunocytochemistry for anti-Pyd and anti-Ref(2)P were all carried out using confocal technology; a Zeiss LSM900 microscope with a 63× Plan-Apochromat 1.4 NA oil objective under Airyscan SR mode. Live-cell fluorescence imaging for the UAS-YFP-Rab5, UAS-YFP-Rab7, UAS-YFP-Rab11, and UAS-mCherry-Atg8a fly strains were all carried out using confocal technology; a Zeiss LSM900 microscope with a 63X Plan-Apochromat 1.4 NA oil objective under Airyscan SR mode. For quantitative comparison of intensities, common settings were chosen to avoid oversaturation. The results are presented as the mean±s.d. Statistical significance: *P<0.05, **P<0.01, ***P<0.001. ImageJ Software Version 1.52a was used for image processing.
Plan apochromat 1.4 n a oil objective
Manufactured by Zeiss
Sourced in Germany
The 63× Plan-Apochromat 1.4 N.A. oil objective is a high-magnification, high-numerical aperture objective lens designed for use with oil immersion microscopy. It provides a magnification of 63× and a numerical aperture of 1.4, which allows for the capture of high-resolution images with a large field of view. The objective lens is made using apochromatic correction, which helps to minimize chromatic aberrations and ensure accurate color reproduction.
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Lab products found in correlation
Lsm900 microscope, by Zeiss (5 mentions)
Plan apochromat 0.8 na air objective, by Zeiss (2 mentions)
Apotome 2 microscope, by Zeiss (2 mentions)
Lsm 880 airyscan confocal microscope, by Zeiss (2 mentions)
Tempmodule system, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Zen 2 blue edition, by Zeiss (1 mentions)
Celltox green, by Promega (1 mentions)
Annexin 5, by BioLegend (1 mentions)
μ slide, by Ibidi (1 mentions)
Lsm800 point scanning confocal microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
17 protocols using plan apochromat 1.4 n a oil objective
1
Microscopic Imaging of Cellular Organelles
2
Indirect Immunofluorescence Microscopy Protocol
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Indirect immunofluorescence was performed as previously described (Spear and Ng, 2005 (link)). In brief, cells were grown to early log phase in selective media, fixed by 3.7% formaldehyde treatment, and permeabilized. Monoclonal mouse anti-HA (1:500), anti-FLAG tag mouse M2 monoclonal antibody (1:500), and rabbit anti-Kar2 (1:1,000) were used as primary antibodies. Mouse anti-Dylight 488 (1:500) and rabbit anti-Dylight 550 (1:500) were used as secondary antibodies. Samples were imaged with a Zeiss LSM 710 confocal microscope with a 100× 1.4 NA oil plan-Apochromat objective (Carl Zeiss MicroImaging). Images were analyzed using ImageJ 1.48v.
3
Live-cell Imaging of Yeast Stress Response
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For live-cell imaging, yeast cells were grown to an exponential phase at 30°C in 3 ml of selective media. Samples were treated with 10 mM DTT for 1 h or inositol depleted for 4 h. Cells undergoing inositol depletion were washed six times before transferring to inositol-free media. 500 μl of cells in selective media were placed on slides coated with 10 mg/ml Concanavalin A (Sigma-Aldrich) mounted onto Attofluor cell chambers (Thermo Fisher Scientific) and imaged at room temperature. Images were captured using Zeiss Axio Observer.Z1 fluorescence microscope (ymNeonGreen) or Zeiss LSM 710 confocal microscope (split Venus) with a 100× 1.4 NA oil plan-Apochromat objective (Carl Zeiss MicroImaging). Images were analyzed using ImageJ 1.48v. Images were deconvoluted using the ImageJ plugin DeconvolutionLab.
4
Automated Epifluorescence Microscopy for Tissue Analysis
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The immunostained ribbons of sections were imaged on an automated epifluorescence microscope (Zeiss AxioImager Z1) using a 63x Plan-Apochromat 1.4 NA oil objective. For each section from the ribbon, an area of 140 μm × 400 μm was imaged and this area encompassed layers 1 through 4. The entire imaged volume was analyzed, but it was subdivided into separate subvolumes corresponding to the different layers. To define the position list for the automated imaging, a custom Python-based graphical user interface, MosaicPlanner (obtained from https://code.google.com/archive/p/smithlabsoftware/ ), was used to automatically find corresponding locations across the serial sections. Images from different imaging sessions were registered using a DAPI stain present in the mounting medium. The images from the serial sections were also aligned using the DAPI signal. Both image registration and alignment were performed with the MultiStackReg plugin in FIJI45 (link).
5
Confocal Microscopy Imaging Protocol
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Confocal imaging was performed with a Zeiss LSM900 microscope using a 63x Plan-Apochromat 1.4 N.A. oil objective under Airyscan SR mode. For quantitative comparisons of fluorescence intensity, common settings were chosen to avoid oversaturation. ImageJ Software was used for image processing.
6
Fluorescence Recovery After Photobleaching
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The treated cells were plated into 35 mm glass bottom confocal dishes (NEST, 801001), and the FRAP experiment was performed on the Zeiss LSM880 Airyscan confocal microscope with a 63x Plan-Apochromat 1.4 NA oil objective. The Zeiss TempModule system was used to control the temperature (37 °C), the humidity and the CO2 (5%) of the imaging system. After imaging for 3 frames, the cells were photo-bleached using 100% laser power with the 488 nm laser (iterations: 50, stop when intensity drops to 50%). The cells were then imaged again every two seconds. The images were analyzed and measured with ZEN 2 blue edition (Zeiss).
7
Visualizing AIM2 Inflammasome Activation in Macrophages
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BMDMs or iBMDMs were seeded 5 × 104/well in eight-well tissue culture–treated μ-Slides (iBidi) or 96-well Cell Culture Microplates, μClear (Greiner Bio-One) overnight and primed the next day with 100 ng/ml LPS 055:B5 for 4 h. The AIM2 inflammasome was activated by transfection of poy(dA:dT) (see the Cell Death Assay section). For time-lapse microscopy, cells were incubated with CellTox Green (Promega) 1:10,000 and AnnexinV (BioLegend) at 500 ng/ml or for mitochondrial health assessment MitoTracker Green and MitoTracker CMXRos were added to OptiMEM at a final concentration of 125 nM. Images were taken every 5 min or every 15 min, respectively. Zeiss LSM800 point scanning confocal microscope equipped with 63× Plan-Apochromat NA 1.4 oil objective, Zeiss ESID detector module, LabTek heating/CO2 chamber, and motorized scanning stage.
8
Visualizing Prrt2 Protein Localization
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HEK293T cells expressing wild-type or mutated Prrt2 proteins were imaged by a confocal microscope (Zeiss LSM700, Germany) with a 100X Plan-Apochromat NA = 1.4 oil objective. Final composite images were processed by the ImageJ software (NIH, USA).
9
Immunostaining of Tubulin and Lipids
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Cells were fixed with 4% paraformaldehyde in PBS (Lonza) for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Alternatively, for immunostaining of glutamylation (GT335) and polyglutamylation (polyE), cells were fixed and permeabilized with cold methanol for 5 min at −20 °C. Then, after blocking with 3% BSA for 30 min, cells were immunostained with polyE antibody at 4 °C overnight, then with polyE and GT335 together at 4 °C overnight, followed by secondary antibody staining at room temperature for 1 h, and finally with anti-α-tubulin Alexa Fluor 647 at room temperature for 2.5 h. For staining of lipid droplets with LD540 dye, cells were incubated with 0.1 μg ml−1 LD540 in PBS for 5 min. Cells were mounted using Fluoromount-G (SouthernBiotech) and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63 × 1.4 NA Plan-Apochromat oil objective (Carl Zeiss). Images were acquired using ZEN software (Carl Zeiss) and processed with ZEN software or Fiji (NIH).
10
Spinning Disk Confocal Microscopy of Live Cells
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Cells were grown on glass-bottom matTek dishes coated with ConA (coverslips treated for ∼2 h with 100 µg/ml ConA in water at room temperature). MatTek dishes were loaded to a Wave FX spinning disk confocal microscope (Quorum Technologies) on an Eclipse Ti microscope (Nikon) equipped with Hamamatsu ImageM EM CCD cameras and Bionomic Controller (20/20 Technology) temperature-controlled stage set to 37°C. After equilibrating to temperature for 10 min, cells were imaged with the 60× 1.4-NA Plan Apo objective (Nikon) using the 403-nm laser and 450/50 filter for BFP, 491-nm and 525/20 for GFP, 561-nm and 593/40 for mStrawberry or mCherry, and 640-nm and 700/60 for mPlum and E2-Crimson. For rapamycin induction, cells were treated with freshly prepared rapamycin (Thermo Fisher Scientific; 10 mM Stock in DMSO, 10 µM final concentration on cells) during imaging.
Airyscan images were acquired on LSM 880 equipped with 63×/1.4-NA plan Apochromat oil objective, using the Airyscan detectors (Zeiss). The Airyscan uses a 32-channel array of GaAsP detectors configured as 0.2 Airy units per channel to collect the data that is subsequently processed using Zen2 software. After equilibrating to 37°C for 30 min, cells were imaged with the 405-nm laser and 450/30 filter for BFP, 488-nm and 525/30 for GFP, 561-nm and 595/25 for mStrawberry or mCherry, and 633-nm and LP 625 for mPlum.
Airyscan images were acquired on LSM 880 equipped with 63×/1.4-NA plan Apochromat oil objective, using the Airyscan detectors (Zeiss). The Airyscan uses a 32-channel array of GaAsP detectors configured as 0.2 Airy units per channel to collect the data that is subsequently processed using Zen2 software. After equilibrating to 37°C for 30 min, cells were imaged with the 405-nm laser and 450/30 filter for BFP, 488-nm and 525/30 for GFP, 561-nm and 595/25 for mStrawberry or mCherry, and 633-nm and LP 625 for mPlum.
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