Yeast cultures were initially grown overnight in synthetic media with 2% galactose. Cultures were then diluted to OD600 of 0.2 and after further growth, cells were harvested at the logarithmic phase (3,000 xg, 5 min, RT). The collected cells were mixed with 1% (w/v) low melting point agarose in a 1:1 (v/v) ratio and were plated on a glass slide. Spinning disk microscope Zeiss Axio Examiner Z1 equipped with a CSU-X1 real-time confocal system (Visitron) and SPOT Flex charge-coupled device camera (Visitron) was used. Samples were observed using Objective Plan-Apochromat 63×/1.4 Oil DIC M27 (Zeiss) at room temperature. Images from three different channels (Brightfield, GFP, and RFP) were acquired using the AxioVision software (Visitron). Cropping and merging of microscope images were done using Fiji software.
Plan apochromat 63 1.4 oil dic m27 objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat 63×/1.4 Oil DIC M27 objective is a high-performance objective lens manufactured by Zeiss. It is designed to provide superior optical performance with a high numerical aperture of 1.4 and a magnification of 63×. The objective is optimized for use with oil immersion and Differential Interference Contrast (DIC) imaging techniques.
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Lab products found in correlation
Elyra ps 1 microscope, by Zeiss (9 mentions)
Lsm 780 confocal microscope, by Zeiss (7 mentions)
Lsm 780, by Zeiss (5 mentions)
Axio zoom v16, by Zeiss (4 mentions)
Pco edge 5.5 camera, by Zeiss (4 mentions)
Fm4 64, by Thermo Fisher Scientific (4 mentions)
Zen 2012, by Zeiss (3 mentions)
Axio observer 7, by Zeiss (3 mentions)
Imaris software, by Oxford Instruments (3 mentions)
880 airyscan microscope, by Zeiss (3 mentions)
Zen software, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Argon laser, by Zeiss (2 mentions)
Lsm 880 super resolution airyscan module system, by Zeiss (2 mentions)
Axio observer, by Zeiss (2 mentions)
Thioflavin t tht, by Merck Group (2 mentions)
Zen black software, by Zeiss (2 mentions)
Elyra 7 axioobserver microscope, by Zeiss (2 mentions)
Pco edge scmos version 4.2 cl hs cameras, by Zeiss (2 mentions)
49 protocols using plan apochromat 63 1.4 oil dic m27 objective
1
Yeast Cultures Microscopy Protocol
2
Localization of Cas13 variants in HEK293 cells
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HEK293 cells were seeded onto poly-D-lysine-coated dishes (35-mm glass bottom, MatTek) and incubated overnight. The following day, the cells were cotransfected with the Cas13 variant-expressing plasmid (pXR-dPspCas13b-NLS-mCherry or pXR-dPspCas13b-NES-mCherry, 300 ng), the gRNA-expressing plasmid (pC016 series, 600 ng), and the luciferase reporter plasmid (psiCHECK2-PTGES3, 10 ng) using TransIT-293 (Mirus) according to the manufacturer’s instructions. The cells were incubated with 0.5 µg/ml Hoechst 33342 (nacalai tesque) for 15 min and observed under an LSM 710 (Carl Zeiss) confocal microscope with an Objective Plan-Apochromat 63×/1.4 Oil DIC M27 (Carl Zeiss).
3
Fluorescence-based Protein-RNA Interaction Assay
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For fluorescence detection, recombinant proteins (FL, polyG, and Cterm) were labeled with fluorescein using the Fluorescein Labeling Kit-NH2 according to the manufacturer’s instructions (Dojindo Molecular Technologies Inc.). CGG99 RNA was labeled with CX rhodamine using the Label IT Nucleic Acid Labeling Kit according to the manufacturer’s instructions (Mirus Bio LLC). Recombinant proteins (9 μM; 8 μM nonlabeled proteins and 1 μM fluorescently labeled proteins) were prepared in 10 mM tris-HCl buffer (pH 7.5) containing 25 mM NaCl, 10 mM MgCl2, and 12% (w/v) glycerol and were then incubated for 1 hour at 23°C. Then, 10% (w/v) 1,6-hexanediol (Sigma-Aldrich) was used to disturb weak interactions that drive LLPS. Fluorescently labeled CGG99 RNA or mTERRA (20 ng/μl) (5′-UUACCGUUACCGUUACCGUUACCG-3′) was prepared in the same buffer. For the preparation of protein/RNA complex solutions, prefolded RNAs with or without 50 μM PpIX were added to the protein solutions, which were then incubated for 1 hour at 23°C. The resultant solutions were covered with coverslips using a 0.12-mm spacer and sealed before measurement. Photobleaching was done with 100% laser power to 50% intensity using the bleaching program of the ZEN software on a Zeiss LSM780 machine. Time-lapse images were recorded every 5 s using a Zeiss Objective Plan-Apochromat 63×/1.4 oil DIC M27 for tracking photorecovery behavior.
4
Microscopic Imaging of Bacterial Pellicles
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Bright field images of whole pellicles were obtained with an Axio Zoom V16 stereomicroscope (Carl Zeiss, Jena, Germany) equipped with a Zeiss CL 9000 LED light source and an AxioCam MRm monochrome camera (Carl Zeiss). The pellicles were also analysed using a confocal laser scanning microscope (LSM 780 equipped with an argon laser, Carl Zeiss) and Plan-Apochromat/1.4 Oil DIC M27 × 63 objective. Fluorescent reporter excitation was performed with the argon laser at 488 nm and the emitted fluorescence was recorded at 484–536 nm and 567–654 nm for GFP and mKate, respectively. To generate pellicle images, Z-stack series with 1 μm steps were acquired. Zen 2012 Software (Carl Zeiss) was used for both stereomicroscopy and CLSM image visualization.
5
Automated Microscopy for Pellicle Imaging
6
Spike Protein Impacts Fibrin(ogen) Clot Formation
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To determine if spike protein causes changes in purified fibrinogen, our purified fibrin(ogen) clot model of choice was fluorescent fibrinogen conjugated to Alexa Fluor™ 488 (Thermo Fisher, F13191). A final fibrinogen concentration of 2 mg.ml−1 was prepared in endotoxin-free water and exposed to 1 ng.ml−1 (final concentrations) spike protein for 30 min at room temperature. A total of 5 μl of the purified fibrinogen was placed on a glass slide, with 2.5 μl of thrombin. The excitation wavelength for our fluorescent fibrinogen model was set at 450–488 nm and the emission at 499–529 nm and processed samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).
7
Platelet Activation Markers in COVID-19 and Long COVID
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The whole blood (WB) (haematocrit) samples of healthy volunteers, COVID-19 and Long COVID/PASC patients were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA). CD62P is found in the granules of platelets and then translocate to the platelet membrane surface. The translocation occurs after the platelet P-selectin is released from the cellular granules during platelet activation [6 (link), 9 (link)]. 4 µL CD62P and PAC-1 was added to 20 µL haematocrit. The haematocrit exposed to the markers was incubated for 30 min (protected from light) at room temperature. The excitation wavelength for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 nm to 570 nm and the emission 577 nm to 607 nm. Processed samples were viewed using the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).
8
Super-resolution Microscopy of Glia Granules
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Super-resolution microscopy was performed with Zeiss LSM 880/super-resolution Airyscan module system with inverted laser scanning confocal microscope AxioObserver and Plan-Apochromat ×63/1.4 Oil DIC M27 objective at Vanderbilt University Cell Imaging Shared Resource (CISR). Images analysis and 3D reconstructions were carried out using Imaris software (version 9.5, Oxford Instruments) and HP Z8 workstation. To measure the percentage of Tud glial granules that overlap with a given protein, Surfaces option in Imaris was used to automatically identify granules and calculate the percentage of particles which contain both proteins. Glial granules (141–287) were analyzed for each Tud/given protein pair.
9
Visualizing Germline Granules in Drosophila
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Microscopy was carried out with Zeiss LSM 880/super-resolution Airyscan module system with inverted laser scanning confocal microscope AxioObserver and Plan-Apochromat 63 ×/1.4 Oil DIC M27 objective at Zeiss Microscopy Customer Center (Thornwood, NY). Images were analyzed and 3D reconstruction was carried out using Imaris software (version 9.3.1, Oxford Instruments) and HP Z8 workstation. For a 3D reconstruction, 7–11 optical sections per a well-defined granule were used using Surfaces option in Imaris. For each granule, Aub and Tud clusters were reconstructed separately and subsequently composite images of the complete granules were generated. For microscopy, preblastoderm and cellular blastoderm embryos expressing functional full-length GFP-Tud20 (link) were immunostained with rabbit anti-GFP antibody (ab290) from Abcam (1:5000) and rat anti-Aub antibody (1:1000)21 (link). This whole-mount immunostaining methodology was described previously42 (link).
10
Microscopic Imaging of Cell Membranes
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Overnight cultures of derivatives of BCBMS14 (JE2 background) expressing sgRNAs against ftsZ (BCBMS16), pbpA (BCBMS17), or murJ (BCBMS18) were each back-diluted 1:1,000 into fresh media containing 10 µg/mL Cm with (induced) or without (non-induced) 100 ng/mL aTc and grown at 37°C to exponential phase (OD600 0.6–0.8). A 1 mL aliquot of each culture was incubated with 2.5 µg/mL Nile Red (membrane stain, Invitrogen) for 5 min at 37°C with shaking. The culture was then pelleted and resuspended in 30 µL PBS. One microliter of cell culture was placed on a thin layer of 1.2% agarose in PBS and imaged via structured illumination microscopy (SIM) using an Elyra PS.1 microscope (Zeiss) with a Plan-Apochromat 63×/1.4 oil DIC M27 objective. SIM images were acquired using five grid rotations, with 34 µm grating period for the 561 nm laser (100 mW), and captured using a Pco.edge 5.5 camera. Images were reconstructed using ZEN software (black edition, 2012, version 8.1.0.484).
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