Islets expressing pAd-RIP1-Epac-SH187-pA were imaged in No. 1.5 glass-bottom dishes on a multiphoton laser scanning system based around a Nikon TE−300 inverted microscope equipped with a 40×/1.15N.A. water immersion objective in a standard imaging solution (135 mM NaCl, 4.8 mM KCl, 5 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH 7.35). Temperature was maintained at 37°C with a Tokai Hit incubator. The Venus fluorescent protein was excited with a Chameleon Ultra laser (Coherent) at 920 nm. Fluorescence emission was captured by a Hamamatsu photomultiplier tube after passing through a 535/70 emission filter (Chroma). The images were collected at 512 × 512 resolution with a 2-µm z-step size at an optical zoom of 1.25 and dwell time of 2 µs.
Hit incubator
Manufactured by Tokai Hit
The Hit incubator is a laboratory equipment designed for controlled temperature and humidity environments. It maintains precise temperature and humidity settings to facilitate cell and tissue culture experiments.
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Lab products found in correlation
2 protocols using hit incubator
1
Multiphoton Imaging of Epac-SH187-Expressing Islets
2
Imaging Mitotic Chromosome Localization
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293A cells were plated on a chambered coverslip (μ-Slide 8 Well Glass Bottom, ibidi #80827) and transfected the next day with 0.8μg plasmid DNA expressing GFP-tagged HoxA11 (p1050), FoxA1 (p1031),46 (link) or Foxc1 (p1104) using 2μl Lipofectamine 2000. The next day, transfection media was replaced with fresh media containing nocodazole at 330nM final concentration. We set up confocal imaging to start at 15 hours after nocodazole treatment and finish by 18 hours. Hoechst 33342 was added just before microscopy. We imaged live cells on a Nikon A1R inverted LUNV confocal laser scanning microscope equipped with a heated stage insert providing CO2 and humidity (Tokai Hit incubator) housed at the Confocal Imaging Core (CIC) at CCHMC. We looked for condensed mitotic chromosomes and captured those images with a 60x oil-immersion objective lens and determined if GFP fusion proteins were colocalized.
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