Functional multicellular Ca2+-imaging was performed on isolated islets previously incubated with Cal-520 acetoxymethyl (AM; 2 µM; Stratech) for 45 min at 37 °C in KRB supplemented with 3 mM glucose. Fluorescent imaging was performed using a Zeiss Axiovert microscope equipped with a 10–20×/0.3–0.5 NA objective, a Hamamatsu image-EM camera coupled to a Nipkow spinning disk head (Yokogawa CSU-10). Volocity software (PerkinElmer Life Sciences) provided the interface. Islets were kept at 37 °C and constantly perfused with KRB-containing 3 mM, 17 mM glucose or 20 mM KCl. For each experiment, ~20 islets were used, isolated from 3 animals per genotype. Imaging data were analyzed with ImageJ software using an in-house macro (available upon request). Fluorescent traces (F) were normalized to baseline recorded under 3 mM glucose (Fmin). The AUC at 17 mM glucose was calculated using different fluorescence baseline values (prior perfusion with high glucose) for each genotype group. The same approach was adopted to measure the AUC during KCl imaging, where baseline values prior to stimulation with KCl were used.
Axiovert microscope
Manufactured by Hamamatsu Photonics
Sourced in United Kingdom, France
The Axiovert microscope is a high-performance inverted microscope developed by Hamamatsu Photonics. It is designed to provide clear and detailed imaging for a variety of applications. The Axiovert microscope features advanced optics and a robust build to deliver reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Cal 520 acetoxymethyl am, by Stratech Scientific (2 mentions)
Csu10, by Yokogawa (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Volocity software, by PerkinElmer (1 mentions)
Diazoxide, by Merck Group (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Plan apochromat 100 1.4 na oil objective, by Zeiss (1 mentions)
Orca r2 cooled, by Hamamatsu Photonics (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti α tubulin, by Abcam (1 mentions)
Rat anti ha, by Merck Group (1 mentions)
6 protocols using axiovert microscope
1
Functional Calcium Imaging of Isolated Islets
2
Calcium Imaging of Pancreatic Islets
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Ca2+ imaging of whole islets was performed after loading with R-GECO (48 h post-isolation) in Krebs-Ringer bicarbonate buffer or cytosolic Cal-520 acetoxymethyl (AM; 2 μmol/l; 24 h post-isolation; Stratech, Cambridge, UK) in modified Krebs-Ringer bicarbonate buffer (140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 2 mmol/l NaHCO3 [saturated with CO2], 1.5 mmol/l CaCl2, 0.5 mmol/l MgSO4, 10 mmol/l HEPES; pH 7.4) containing 3 mmol/l or 17 mmol/l glucose, 17 mmol/l glucose with 0.1 mmol/l diazoxide (Sigma-Aldrich, Dorset, UK), or 20 mmol/l KCl. Images were captured at 0.5 Hz on a Zeiss Axiovert microscope equipped with a ×10 0.3–0.5 NA objective, a Hamamatsu image-EM camera coupled to a Nipkow spinning-disk head (Yokogawa CSU-10; Runcorn, UK) and illuminated at 490 nm or 530 nm. Data were analysed using ImageJ (https://imagej.nih.gov/ij/download.html , accessed 15 February 2020) with a purpose-designed macro (available upon request).
3
Lipid Staining in Cryosections
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Cryosections on glass slides were fixed with 10% (v/v) formaldehyde in PBS for 1 h at 23 °C, rinsed twice with water and then stained with 0.1% (w/v) Oil Red O in 75% (v/v) isopropanol at 23 °C. After 2 h, the stained cryosections on glass slides were rinsed twice with water to remove unincorporated dye and photographed using a Zeiss AxioVert microscope with phase contrast optics and Hamamatsu digital/video camera.
4
Immunostaining of Membrane Fractions
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Sonicated membrane fractions were applied on degreased air-dried coverslips and fixed with fixative buffer (2.5% PFA in fractionation buffer) at room temperature for 10 min. Fractions were washed and blocked in blocking buffer for 5 min. Antibodies were placed in blocking buffer and centrifuged briefly at 135,000 rpm. Primary antibodies (including TMR-Star coupled SNAP ligand; New England Biolabs, Ipswich, MA) were incubated on the samples for 15 min, followed by 3× washes for a total of 10 min in fractionation buffer. Secondary antibodies (Alexa Fluor 488 and 647, Jackson Immunoresearch, West Grove, PA; CY3, Molecular Probes) were incubated on the samples for 15 min, followed by the same washing step. Samples were postfixed in fixative buffer for 5 min, washed 1× briefly, and mounted with Mowiol on degreased, air-dried glass slides. Samples were imaged on a Zeiss Axiovert microscope equipped with a Hamamatsu Orca-R2 cooled digital CCD camera using 1 × 1 binning (pixel size, 65 nm), 12-bit pixel depth, and a Zeiss Plan-Apochromat 100×/1.4 NA oil objective.
5
Measuring Cytosolic Calcium Dynamics
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Cytosolic Ca2+ concentration ((Ca2+)cyt) was monitored as reported previously [22 (link)] by fluorescence imaging of cells using an inverted Zeiss Axiovert microscope equipped with a OrcaER Hamamatsu digital camera (Hamamatsu, Photonics, France). Cells were loaded with fura-2/AM (4 µM, 60 min) in external saline solution containing (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, glucose 10, Hepes/Na 10 (pH 7.42). For SOCE, cells were washed twice and treated with CPA or thapsigargin (1 µM, 10 min) in the same medium except that it was devoid of Ca2+ and contained also 0.5 mM EGTA. Then cells were located in the stage of an inverted microscope and subjected to fluorescence imaging whereas continuously perfused with external medium at 37 °C. Cells are epi-illuminated alternately at 340 and 380 nm using band pass filters and light emitted above 520 nm at both excitation lights was filtered by the dichroic mirror, collected every 5 to 10 s with a 40×, 1.4 NA, oil objective.
6
Overexpression of Tubb4a in RPE Cells
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C-terminal HA-tagged WT or Tubb4aJit cDNA was cloned into the pcDNA 3.1 vector and transfected into retinal pigmented epithelial cell (RPE). RPE cells (1 × 105) were seeded onto coverslips in a 24-well plate. Two micrograms of plasmid DNA was used to transfect cells (Lipofectamine 3000, Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were fixed for 15 min with methanol 48 hours posttransfection and blocked with PBS with 0.1% Triton X-100 and 1% normal goat serum for 1 hour. The cells were incubated with the primary antibodies [rabbit anti–α-tubulin (Abcam) and rat anti-HA (Sigma-Aldrich)] overnight at 4°C, rinsed, and incubated with secondary antibodies [Alexa Fluor 488 donkey anti-rabbit (Life Technologies) and Alexa Fluor 594 goat anti-rat antibodies (Life Technologies)] for 1 hour at room temperature. Images were obtained on a Zeiss Axiovert microscope using a Hamamatsu Oraca Flash 4.0 camera driven by Zen software.
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