Fresh pancreas samples were treated with 30% sucrose overnight, embedded in OCT, and stored at −80°C. For staining, 10 µm sections were cut and fixed in 3.7% formaldehyde for 1 hour. Sections were permeabilized with 0.5% Triton X-100/PBS for 5 minutes, and stained with insulin primary antibody (Sigma I2018, 1:10,000, 30 min), washed, and then secondary antibody (Abcam ab150117, 1:2,000, 30 min). Following rinses with 1x PBS, the ORO working solution was applied for 30 minutes. The sections were then stained with DAPI (ThermoFisher, D1306) in 1% BSA for 1 minute (adapted from Koopman et al.).49 (link) Samples were imaged using a Zeiss Axiovert 200M microscope and an Olympus BX51TF microscope. Image quantification for Islet-LC was performed in ImageJ by tracing the Ins+ area and measuring the ORO-positive area within each islet. Acinar lipid content quantification was performed by tracing the Ins-negative area surrounding the islets. Islet-LC was measured for 19–38 islets per donor. Acinar lipid content was measured on 7–15 images per donor.
Axiovert 200m microscope
Manufactured by Olympus
The Axiovert 200M is an inverted research microscope designed for a wide range of applications in cell biology, developmental biology, and materials science. It features a motorized focusing drive, a high-quality optical system, and an ergonomic design. The Axiovert 200M provides precise control and high-resolution imaging capabilities for advanced research and analysis.
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2 protocols using axiovert 200m microscope
1
Pancreatic Islet Lipid Profiling
2
Immunofluorescent Staining of Cells and Nucleoplasts
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Cells were fixed with 4% paraformaldehyde in Krebs S-buffer and permeabilized in 0.2% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked for 30 min in PBS containing 5% BSA. Primary antibodies in PBS containing 1% BSA were stained overnight at 4°C followed by extensive washes in PBS. Dyes such as ER-Tracker Red (BODIPY), MitoTracker Green FM, and calcein-AM require living cells for staining and were used per the manufacturer’s recommendation. Fluorescent dye–conjugated secondary antibodies were diluted to 1:1,000–1:3,000 in 1% BSA in PBS and applied for 1 h at ambient temperature followed by extensive washes in PBS. For nucleoplast stains, nucleoplasts were seeded onto 20 µg/ml FN–coated glass coverslips and, when appropriate, fixed after 30 min. Glass coverslips coated with poly-l -lysine resulted in higher retention of nucleoplasts. Nucleoplasts were permeabilized and stained as described above. Fluoromount-G (Electron Microscopy Sciences) was used as the mounting medium for fixed cells on coverslips. Fluorescent images were acquired on either a Zeiss Axiovert 200M microscope using 20× or 40× objectives or on an Olympus FV1000 using a 40× objective.
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