RAW 264.7 macrophages (0.2 x 106 cells) were seeded on glass coverslips in a 24 well plate and left for adhesion overnight. Cells were then incubated with nanoemulsion (30 μL) in culture media (1 mL) for two hours at 37 °C. Cell were then washed with DPBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and stained with DAPI for nuclei visualization. For kinetic studies, cells were incubated with nanoemulsion (30 μL in 1 mL medium) for 5, 15, 30 and 60 min at 4 and 37 °C, fixed with 4% PFA, and stained with DAPI. After washing with DPBS, coverslips were mounted using Diamond anti-fading medium (Invitrogen, Grand Island, NY). Fluorescence was monitored using a Zeiss Apotome system equipped with a Zeiss HPO PL APO 63x oil immersion lens (numerical aperture 1.4-0.6). For colocalization with acidic compartments, cells were seeded on a glass bottom dishes and left for adhesion. Cells were then incubated with nanoemulsion (30 μL in 1 mL medium) for 45 min at 37 °C, and Lysotracker Green DND-26 (1 μM) (Invitrogen, Grand Island, NY) was added for 15 min at 37 °C. Cells were washed with DPBS and immediately imaged using a Zeiss Apotome system equipped with a Zeiss HPO PL APO 63x oil immersion lens (numerical aperture 1.4-0.6).
Apotome system
Manufactured by Zeiss
Sourced in Germany
The ApoTome system is an imaging solution developed by Zeiss. It provides optical sectioning capabilities, allowing users to acquire high-quality, artifact-free images by reducing out-of-focus light. The ApoTome system is designed to enhance the performance of fluorescence microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m2, by Zeiss (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (12 mentions)
Axiovision software, by Zeiss (11 mentions)
Axiovert 200 inverted microscope, by Zeiss (7 mentions)
Axiovision 4, by Zeiss (6 mentions)
Axio imager z1 microscope, by Zeiss (5 mentions)
Axio observer inverted microscope, by Zeiss (5 mentions)
Zen software, by Zeiss (5 mentions)
Axio observer inverted fluorescence microscope, by Zeiss (5 mentions)
Axiocam mrm camera, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Axiovert 200m microscope, by Zeiss (4 mentions)
Alexa labeled secondary antibody, by Thermo Fisher Scientific (3 mentions)
Zen 2012, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Hpo pl apo 63x oil immersion lens, by Zeiss (3 mentions)
Goat α rabbit igg texas red, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa 488 green, by Thermo Fisher Scientific (2 mentions)
Alexa 568 red, by Thermo Fisher Scientific (2 mentions)
97 protocols using apotome system
1
Macrophage Uptake Kinetics of Nanoemulsions
2
Orientation of Limb Ectoderm Cells
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Embryonic limbs were dissected in PBS, collected in 4% PFA and fixed for 12–16 h depending on the age, washed in PBS and cryoprotected. Cryostatic sections (12–15 μm thick) were collected on glass slides, blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT and incubated with the following primary antibodies, diluted from 1:250 to 1:50 in 1% BSA in PBS, ON at 4°C: anti-GM130 (BD), with anti-p63 (4A4 sc-8431, Santa Cruz), anti ZO-1 (InVitrogen) and with anti-E-cadherin (36/E 610182, BD). Sections were rinsed with PBS and incubated with secondary antibodies anti-mouse-Cy2 and anti-rabbit-Cy3 (Jackson ImmunoResearch) diluted 1:200, 1 h at RT, rinsed in PBS, counterstained with DAPI and examined with a Zeiss Observer-Z1 fluorescent microscope, equipped with the Apotome system.
For a semi-quantitative assessment of the orientation of the AER cells in whole-limbs, the results were visualized using the Rose2.0 software, showing the angle of each cell's longest axis as a unidirectional rosette graph divided into Bins of 15°. Each interval represents 25 cells per Bin. A minimum of N = 150 AER cells were counted for each of the two genotypes.
For a semi-quantitative assessment of the orientation of the AER cells in whole-limbs, the results were visualized using the Rose2.0 software, showing the angle of each cell's longest axis as a unidirectional rosette graph divided into Bins of 15°. Each interval represents 25 cells per Bin. A minimum of N = 150 AER cells were counted for each of the two genotypes.
3
Visualizing DNA Synthesis in Cells
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To visualize individual cells synthesizing DNA we used the Click–It kit (Invitrogen) according to manufacturer’s instructions. This kit allows for robust statistical analysis in small populations of cells while utilizing the highly-specific labeling methodology of BrdU or [3H]-thymidine incorporation. Briefly, subconfluent cells were grown in 96-well plates in quadruplicate, starved, and transferred to complete media for 16 h. Then EdU (10 μM) was incorporated for 4 h and cells were fixed in a 4% paraformaldehide/PBS solution (Electron Microscopy Sciences, PA USA). Samples were counterstained with Hoescht 33342 and visualized under Axio Imager Z1 microscope equipped with ApoTome system controlled by AxioVision software (Carl Zeiss).
4
Immunostaining of PDCD10 in Tissue Sections
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Immunostaining of laminin was performed as described previously [27 (link)]. For immunofluorescent staining of PDCD10, sections were incubated with rabbit anti-PDCD10 (Atlas, 1:65) at 4 °C overnight and then incubated with biotinylated goat anti-rabbit IgG (Dako, 1:400) at 37 °C for 1 h followed by the substrate reaction with FITC-labelled avidin (Dako, 1:400) at room temperature for 1 h. For double-immunofluorecent staining, the following antibody mixtures were applied to the sections: rabbit anti-PDCD10 (Atlas, 1:65) and mouse anti-GFAP (Sigma, 1:200); rabbit anti-PDCD10 and mouse anti-CD31 (Dako, 1:20); rabbit anti-PDCD10 and mouse anti-CD68 (Dako, 1:100); rabbit anti-PDCD10 and mouse anti-PCNA (Dako, 1:200); mouse anti-PDCD10 (Santa Cruze, 1:50) and rabbit anti-caspase 3 (active form) (Cell signaling, 1:400). After incubation overnight, the sections were incubated with the mixture of biotinylated goat anti-rabbit IgG (1:400) and Texas red anti-mouse IgG (H + L) (Vector Laboratories, 1:200) followed by the substrate reaction with FITC-labelled avidin. Counterstaining was performed with Hoechst-33258. The sections were finally analyzed by using a fluorescence microscope with ApoTome System (Zeiss, Axio Imager M2).
5
Immunofluorescent Detection of MDV Antigens
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Detection of lytic MDV antigens was performed on 7 µm cryosections of bursa and thymus. Sections were fixed in 4% paraformaldehyde (PFA), permeabilized and blocked in a PBS solution containing 10% BSA and 0.1% Triton. Cryosections were next incubated overnight at 4 °C with an antibody cocktail mix directed against three MDV antigens (ICP4, VP22 and gB) diluted (1:1000 each) in a PBS solution containing 1% BSA, 0.1% Triton. Replicate sections were treated under identical conditions with the diluent only, to serve as negative controls. After PBS washes, cryosections were then incubated 45 min with a secondary antibody, GAM-Alexa 488 (IgG) (1:2000) for the bursa and GAM-Alexa 488 F(ab’)2 (1:500) for the thymus. After PBS washes, cryosections were stained with Hoechst-33342 (1:2000) for 1 min and mounted with Mowiol® 4–88 (Calbiochem). All cryosections were observed under an Axiovert 200 M inverted epi-fluorescence microscope equipped with the ApoTome system (Zeiss). Images were captured with a CCD Axiocam MRm camera (Zeiss) using the Axiovision software (Zeiss).
6
Immunofluorescence Microscopy of Tight Junctions
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Caco-2 cells grown until differentiation on Lab-Tek chamber slides (Nunc, Merck Millipore, Darmstadt, Germany) were treated for 3 h with 1 mg/mL PT-gliadin or PT-gliadin+bact in complete medium, then were washed with PBS and fixed with 2% formaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1% triton, exposed to blocking buffer (5% BSA in PBS) for 30 min, and then incubated for 2 h at room temperature with rabbit polyclonal anti-zonulin (ZO-1) (1:100 dilution) or anti-occludin (1:100) antibody (Thermo Fisher, Waltham, MA, USA) in 1% BSA in PBS. After washing, chambers were exposed for 1 h to the secondary goat anti-rabbit antibody Alexafluor 488 (1:100 dilution) (Abcam, Cambridge, UK). Nuclei were stained for 1 min with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in saline solution, and the coverslips were mounted with Mowiol. Fluorescence signal was analyzed by recording stained images using an AxioObserver inverted microscope, equipped with the ApoTome System (Carl Zeiss Inc., Oberkochen, Germany). Microscopy imaging was performed using the Axiovision software (Zeiss).
7
Embryonic DiI Labeling in Coelomic Cavity
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DiI (Invitrogen) was dissolved in ethanol (0.1%), heated at 45 °C for 3 min and diluted 1:10 with 0.3 M sucrose. This solution was injected into the coelom of the right side of E2.0 embryos using a glass capillary. Thereafter, the embryos were incubated for various time periods, killed, fixed overnight at 4 °C in PBS containing 4% paraformaldehyde (PFA) and sectioned by cryostat at 10-μm thick on Platinum Pro-coated glass slides (Matsunami). Images were obtained on an AxioPlanII microscope with the Apotome system (Carl Zeiss).
8
Isolation and Sorting of Retinal Nuclei
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For each experiment, retinas from two to four HRGP-Cre; R26-CAG-LSL-Sun1-sfGFP-myc mice were homogenized using a loose pestle in a Dounce homogenizer in ice-cold homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine) with EDTA-free protease inhibitor (11 836 170 001, Roche, Basel, Switzerland). After addition of 5% IGEPAL-630 to bring the sample to 0.3% IGEPAL-630, the sample was further homogenized using a tight pestle. The sample was filtered using a 40 µm strainer (08-771-1, Fisher Scientific, Waltham, MA), mixed with 1.5 ml of 50% iodixanol density medium (D1556, Sigma, St. Louis, MO), and pelleted by centrifugation at 10,000g for 18 min in a swinging bucket centrifuge at 4°C. Nuclei were sorted using a MoFlo MLS high-speed cell sorter (Beckman Coulter, Brea, CA). Nuclei were sorted into either Buffer RLT for RNA preparation or PBS for DNA preparation (see below). An aliquot of nuclei sorted using the same parameters was placed into an additional tube. After sorting, this aliquot was inspected using a Zeiss Imager Z1 and Apotome system in order to verify the purity of the sorted sample.
9
Quantifying EV Uptake in mTEC
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To trace EVs by fluorescence microscopy, MSCs were labeled with Vybrant Cell Tracers Dil and Syto-RNA (Life Technologies, Carlsbad, CA) as previous described [20 (link)]. EVs obtained from labeled cells were concentrated and subjected to gradient separation as described above. For EV incorporation, mTEC were plated in 24-well plated and treated with different doses of labeled cCM-EVs (50,000, 150,000, 300,000 or 600,000 EVs/cell) for 24 h. Quantitative analysis of the EV uptake was conducted by FACS. To determine the incorporation of each fraction, mTEC were seeded into 24-well plates and incubated with labeled EVs (150,000 EVs/cell) from the different combined fractions for 24 h. The up-take of EVs was analyzed by microscope analysis using the ApoTome system (Carl Zeiss, Oberkochen, Germany). Hoechst 33,258 dye (Sigma-Aldrich) was added for nuclear staining.
10
Immunofluorescence Analysis of NF-κB Activation
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Subconfluent HaCaT and HFs grown on coverslips were treated with rNPM. Cells were fixed 30 min with 4% paraformaldehyde in PBS and permeabilized for 5 min with 0.1% Triton X-100. The following primary antibodies diluted in 1% BSA were incubated for 1 h at 37°C in a humid chamber: α-p65rel NF-kB (1:200; F6 Santa Cruz). Mouse IgG isotype control (Santa Cruz) was used at the same concentration of primary antibody. After PBS washes, secondary antibodies goat α-rabbit IgG-Texas Red (1:200 in PBS; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were added and incubated for 1 h at 37°C in a humid chamber. Nuclei were stained with DAPI (1:10,000 in PBS; Sigma). Images were acquired with the ApoTome System (Zeiss) connected with an Axiovert200 inverted microscope (Zeiss); image analysis was performed with ZEN software (Zeiss).
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