Cells were plated on a 24-well μ-Plate (Ibidi®). The medium was changed to CO2 Independent Medium (Gibco®) 6 h before filming. DNA was stained by addition of the SiR-Hoechst-647 Dye (Spirochrome) to the medium 1 h before imaging. Cells were imaged every 5–10 min in a heated chamber (37 °C) on a 3i MarianasTM system (Intelligent Imaging Innovations, Inc.) equipped with Axio Observer Z1 microscope (Zeiss), Plan-Apochromat ×40/1.4NA oil objective, M27 with DIC III Prism (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu), and controlled by Slidebook Software 6.0 (Intelligent Imaging Innovations, Inc).
Plan apochromat 40 1.4na oil objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat ×40/1.4NA oil objective is a high-performance microscope objective lens manufactured by Zeiss. It is designed to provide a large numerical aperture of 1.4, which enables high-resolution imaging and enhanced light-gathering capabilities. The lens is also plan-apochromatic, ensuring accurate, flat-field imaging across the entire field of view.
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Lab products found in correlation
Axio observer z1 microscope, by Zeiss (2 mentions)
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (2 mentions)
Co2 independent medium, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
μ plate, by Ibidi (1 mentions)
Dic 3 prism, by Zeiss (1 mentions)
24 well plate, by Ibidi (1 mentions)
Plan apochromat 63 1.4 na oil objective, by Zeiss (1 mentions)
Lsm 900 airyscan 2 microscope, by Zeiss (1 mentions)
Lsm 880 airyscan microscope, by Zeiss (1 mentions)
Lsm 900 airyscan 2, by Zeiss (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Lsm 700 confocal system, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti rfp primary antibody, by Rockland Immunochemicals (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (1 mentions)
4 protocols using plan apochromat 40 1.4na oil objective
1
Live-cell imaging of DNA dynamics
2
Live-cell imaging of GFP-BubR1 dynamics
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Cells were plated on a 24-well µ-Plate (Ibidi, Martinsried, Germany). Drugs were diluted in CO2 Independent Medium (Gibco) and added to the cells 1 hr before filming. Cells were imaged every 20 to 30 min in a heated chamber (37°C) on a 3i Marianas system (Intelligent Imaging Innovations Inc., Göttingen, Germany) equipped with Axio Observer Z1 microscope (Zeiss), Plan-Apochromat 40×/1.4NA oil objective, M27 with DIC III Prism (Zeiss, Oberkochen, Germany), Orca Flash 4.0 sCMOS Camera (Hamamatsu, Hamamatsu City, Japan) and controlled by Slidebook Software 5.5 (Intelligent Imaging Innovations Inc). For cells expressing the GFP-BubR1 proteins, only cells in which kinetochores were visible were considered for the analysis.
3
Quantification of Synaptic Contacts on Tanycytes
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Confocal micrographs were acquired on Zeiss LSM710, LSM880/Airyscan or Zeiss LSM900/Airyscan 2 setups. We used a Zeiss AXIO Observer ApoTome.2 platform for epifluorescence microscopy. The number of VGLUT2+ presynapses contacting vimentin+ tanycytes were determined by using a Zeiss LSM880/Airyscan microscope equipped with a Plan-Apochromat 63×/1.4 NA oil objective (Zeiss). We separately acquired 2 × 2 tile scans covering each tanycyte subcategory in coronal brain sections at both −1.94 mm and −2.30 mm relative to bregma. Orthogonal z-stacks were acquired at a depth of 25 µm. Images to quantify the intensity of pERK1/2 were captured on an LSM880 microscope equipped with a Plan-Apochromat 25×/0.8 Imm Korr DIC M27 objective (Zeiss). Images showing complementary GluA2 and VGLUT2 signals within individual synapses were captured on a Zeiss LSM900/Airyscan 2 microscope equipped with a Plan-Apochromat 40×/1.4 NA oil objective.
4
RNA Expression Profiling in Muscle Stem Cells
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In situ hybridization was performed using the RNAscope Multiplex Fluorescent Reagent kit v2 (Advanced Cell Diagnostics) following manufacturer's protocol with some modifications. TA from Pax7CRE-ER;ROSA26tdTomato mice was dissected and fixed in 2% PFA for 2 h at 4 ºC, frozen and processed into 10 μm longitudinal-sections. Slices were digested with Protease Plus. Probe hybridization was performed by incubation of mRNA target probes for 2 h at 40 °C. Slices were hybridized with two probe-sets of dual probes using Cd34-C3 as common probe in each set and Myog-C2 or Notch3-C2 as companion probes. Cd34-C3 probe was conjugated to Opal 690 fluorophore while Myog-C2 and Notch3-C2 were conjugated to Opal 570 fluorophore (Akoya Biosystems). For SC immunofluorescence, sections were blocked in 3% BSA for 1 h at room-temperature prior to incubation overnight with anti-RFP primary antibody (Rockland, 600-401-379, 1:200) at 4 ºC. Anti-rabbit Alexa Fluor 488 secondary antibody (A11008, Life Technologies, 1:500) and DAPI (Invitrogen) were added for 1 h at room-temperature. After washing, sections were mounted with Prolong Gold Antifade Mountant (Thermo Fisher). Confocal images were taken with Zeiss LSM-700 confocalsystem with a Plan-Apochromat 40 × /1.4 NA oil objective.
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