Fluorescent dyes (488, 555, 594, or 647) conjugated to wheat germ agglutinin (WGA)71 (link) were sterotaxically injected into layer 1 within A1 of adult C57Bl6/J mice (>P60; 3 male and 3 female). Four superficial (~50μm below pial surface) injections were made within the left hemisphere of A1 (100nL/injection site) along the rostral-caudal tonotopic axis (2.2mm–3.5mm posterior to Bregma) using glass pipettes pulled with a DMZ micropipette puller attached to a 5μL Hamilton syringe with a motorized piston (speed ~ 25nL/min). The head wound was sutured, mice were immediately administered an injection of meloxicam, and recovered on a heating pad with accessible food pellets and HydroGel. The mice were returned to their home cages, and 24 h later were were perfused intracardially with saline followed by 4% PFA in 0.1M PB and then post-fixed overnight at 4oC. Brains were washed in PBS, and thalamocortical slices (100 μm) were sectioned on a vibrating microtome (Leica Microsystems, VT1200S). Sections were incubated for 2 h at RT in 1:200 NeuroTrace 435/455 (Life Technologies) in PBS-T, and mounted using ProLong Gold (Life Technologies). Images were acquired on a Zeiss LSM 710 confocal using a 40x, 1.3NA oil immersion PlanAPO objective (1.1x zoom).
Plan apo objective
Manufactured by Zeiss
Sourced in Germany
The Plan Apo objective is a high-performance microscope objective designed by ZEISS. It provides excellent image quality and optical performance across a wide field of view. The objective is optimized for flat field imaging and delivers consistent resolution and contrast throughout the entire field of view.
Automatically generated - may contain errors
Lab products found in correlation
Vt1200, by Leica (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Neurotrace 435 455, by Thermo Fisher Scientific (2 mentions)
Lsm 710 confocal, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Sp600125 pan jnk inhibitor, by Enzo Life Sciences (2 mentions)
C apochromat 1.2 w m27 objective, by Zeiss (2 mentions)
710 confocal microscope, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (1 mentions)
X cite 110led, by Excelitas (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Tetraspeck microsphere, by Thermo Fisher Scientific (1 mentions)
Lsm780 elyra ps1 microscope, by Zeiss (1 mentions)
Ixon 887 1 k camera, by Zeiss (1 mentions)
1.518 refractive index oil, by Zeiss (1 mentions)
Fluoromont g, by Thermo Fisher Scientific (1 mentions)
Zen 2, by Zeiss (1 mentions)
Lsm800 point scanning confocal microscope, by Zeiss (1 mentions)
Definite focus 2 system, by Zeiss (1 mentions)
Lsm510 meta nlo, by Zeiss (1 mentions)
12 protocols using plan apo objective
1
Fluorescent Tracing of Auditory Cortex
2
Inverted Epifluorescence Microscopy Imaging
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Fixed HMEC-1 cells were imaged with a Zeiss (Axiovert 200M) inverted epifluorescence microscope fitted with a 63× (1.4 NA) Plan Apo objective (Carl Zeiss) using a CCD camera (Orca ER, Hamamatsu). The sample was illuminated using a broadband LED illumination (X-Cite 110LED, Excelitas Technologies) filtered through a standard Cy3 filterset (Cy3-4040C-ZHE M327122 Brightline, Semrock). Signal from the sample was also filtered through this filterset before being acquired by the camera.
3
Super-Resolution Imaging of Fibroblasts
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IMR-90 fibroblasts at PN16 and PN35 were cultured on precision glass coverslips thickness No. 1.5 H (Marienfeld Superior). Immunofluorescence was performed as indicated in the previous section, using Fluoromont-G (Thermo fisher Scientific) as mounting medium. SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using 63×/1.4 oil Plan Apo objective with a 1.518 refractive index oil (Carl Zeiss) and an EMCCD Andor Ixon 887 1 K camera for the detection. Fifteen images per plane per channel (five phases, three angles) were acquired with a Z-distance of 0.09 μm to perform 3D-SIM images. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same conditions as the sample. The SIMcheck plugin in imageJ was used to analyze the acquisition and the processing in order to optimize for resolution, signal to noise ratio, and reconstruction pattern65 (link).
4
Live Imaging of Cellular Structures
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All in vitro imaging experiments were performed using LSM800 point-scanning confocal microscope (Zeiss) equipped with 20× air immersion objective (for sphere imaging) and 63 × oil immersion Plan Apo objective (for all other experiments), as well as 405, 488, 563, and 630 nm lasers. Temperature, CO2, and humidity were controlled throughout live imaging using an automated temperature control system and a gas mixer. For time-lapse imaging, Zeiss Definite Focus.2 system was used to ensure image stabilization. Images were acquired using Zen 2 software (Zeiss) at 16-bit depth, and the acquisition settings were kept constant for the same type of experiments to aid statistical analysis.
5
Corneal SHG Imaging and Analysis
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A 160 µm wide nasal-to-temporal section was cut from one of the strips from sample 3L using a sledge microtome (HM440E; Microm, Walldorf, Germany). The section was then immediately covered by 1 : 1 PBS glycerol solution and sealed between a 1 mm microscope glass slide and a 0.16 mm coverslip. SHG images were acquired with an LSM 510 META NLO instrument (Carl Zeiss, Cambridge, UK) and 20× 0.8NA Plan Apo objective. Illumination was typically about 3 mW in approximately 140 fs pulses at 800 nm from a Chameleon Ultrafast laser (Coherent Scotland Ltd, Glasgow, UK). SHG contrast images were collected in the forward propagating direction at 400 ± 10 nm through a high-extinction near-infrared blocking filter HQ400/20m-2p (Chroma Technology Corp., VT. USA). Representative optical sections through the physical corneal slices, away from the cut surfaces, were collected using a simple tiling procedure with an automated stage but without any image processing, edge adjustments or other data manipulation. The pixel spacing in the SHG images was 0.77 µm.
6
Visualizing Subcellular Sphingomyelin Localization
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Immunofluorescence microscopy was performed as described previously (Shiomi et al., 2015 (link)). In brief, cells cultured on coverslips were fixed with 3% formalin prepared in PBS for 10 min at RT, treated with 0.4% Triton X-100 prepared in PBS for 5 min, and washed with PBS. Fixed cells were blocked with 5% BSA prepared in PBS for 30 min at RT. Antibodies were diluted in this blocking solution. Cells were incubated with primary antibodies for 1 h at RT and with secondary antibodies for 30 min at RT. Specimens were observed at RT with a confocal microscope (LSM700; Carl Zeiss MicroImaging, Inc.) equipped with a Plan-APO objective (63/1.40 N.A. oil-immersion) with appropriate binning of pixels and exposure times. Images were analyzed with ZEN 2012 (Carl Zeiss MicroImaging, Inc.). To visualize the subcellular localization of sphingomyelin, living cells were stained with His-RFP-Lysenin or His-GFP-Lysenin prepared in serum-free DMEM and observed after washing out unbound protein with serum-free DMEM. NucBlue Live Ready Probes (R37605; Thermo Fisher Scientific) was used to stain the nuclei of live cells. All living cells were observed on a stage heater at 37°C or 20°C (Fig. S3 C ).
7
Fluorescent Tracing of Auditory Cortex
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Fluorescent dyes (488, 555, 594, or 647) conjugated to wheat germ agglutinin (WGA)71 (link) were sterotaxically injected into layer 1 within A1 of adult C57Bl6/J mice (>P60; 3 male and 3 female). Four superficial (~50μm below pial surface) injections were made within the left hemisphere of A1 (100nL/injection site) along the rostral-caudal tonotopic axis (2.2mm–3.5mm posterior to Bregma) using glass pipettes pulled with a DMZ micropipette puller attached to a 5μL Hamilton syringe with a motorized piston (speed ~ 25nL/min). The head wound was sutured, mice were immediately administered an injection of meloxicam, and recovered on a heating pad with accessible food pellets and HydroGel. The mice were returned to their home cages, and 24 h later were were perfused intracardially with saline followed by 4% PFA in 0.1M PB and then post-fixed overnight at 4oC. Brains were washed in PBS, and thalamocortical slices (100 μm) were sectioned on a vibrating microtome (Leica Microsystems, VT1200S). Sections were incubated for 2 h at RT in 1:200 NeuroTrace 435/455 (Life Technologies) in PBS-T, and mounted using ProLong Gold (Life Technologies). Images were acquired on a Zeiss LSM 710 confocal using a 40x, 1.3NA oil immersion PlanAPO objective (1.1x zoom).
8
Confocal Imaging of Fluorescent Specimens
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Confocal images of fluorescent specimens were obtained with a Nikon A1 confocal microscope equipped with a 20 × 0.75 numeral aperture (NA) Plan Apo objective and a 60 × 1.49 NA oil immersion objective or a Carl Zeiss LSM 900 confocal microscope equipped with the laser module URGB (diode laser 405 nm; diode laser 488 nm; diode (SHG) laser 561 nm and diode laser 640 nm) and the Airyscan 2. Plan-Apochromat 20x (0.8 NA) objective. Confocal images were captured with a distance interval of 0.5 µm between z-sections. The xy view of the confocal images is presented as maximal projections of z stacks, and xz or yz slice views of the regions of interest were reconstructed to illustrate the protein colocalization. For cell culture, images were acquired by a single focal plane. All confocal images were captured and processed using Nikon NIS-Elements AR (v.4.6) or ZEN 3.0 (Carl Zeiss), whereas brightness, contrast and gamma correction were performed if necessary. Confocal images with Z-stacks were utilized for 3D reconstruction using Imaris 9.7 (Oxford Instruments) (debris engulfment) or ZEN 3.0 (Carl Zeiss) (whole-mount retina).
9
Screening Inhibitor Effects on Intracellular Trafficking
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Inhibitor library was obtained from Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology. EpH4 cells expressing SS-GFP-Lys are seeded at 1.0 × 104 cells/well on a 96 well glass-bottom plate, and after 12 h of incubation, each inhibitor is added to a final concentration of 20 μM. After treatment with each inhibitor for 24 h, the medium was changed to Hepes-buffered saline and the cells were observed at 37°C with a confocal microscope (LSM700; Carl Zeiss MicroImaging, Inc.) equipped with a Plan-APO objective (63/1.40 N.A. oil-immersion).
10
Real-Time Imaging of JNK Inhibitor Effects
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Cultures were treated with pre-warmed 37°C serum-free media containing a 1:1000 dilution of DMSO for vehicle control or 20 μm SP600125 pan-JNK inhibitor (Enzo Life Sciences BML-EI305-0010) and immediately transferred to a Zeiss 710 Confocal Microscope with stable environmental controls maintained at 37°C with 5% humidified CO2. Multiposition time-lapse z-series were acquired at 10-min intervals over a 12-h period with a 20× Plan-Apo objective (Zeiss) for overall migration analysis, nucleokinesis distance, and swelling distance measurements. For measurements requiring higher temporal and spatial resolution, such as swelling duration, branch dynamics, and visualization of subcellular structures in electroporated cells, cultures were imaged using multiposition time-lapse z-series at 2- to 2.5-min intervals over a 4- to 10-h period with a 40× C-apochromat 1.2-W M27 objective (Zeiss).
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