Microchannels (containing axons that connect the two hippocampal slices together) were selected for imaging and analysis based on the number of axons within a channel, and provided that there was no glial cell migration down the microchannel. Images were taken using a computer interfaced inverted Olympus IX81 DSU microscope (Olympus) controlled by Slidebook software (Olympus) using a 40X objective. Only the area within the pressurized section was imaged and analyzed. Each microchannel was split into 11 sections (the number of 40X objective images it took to traverse the pressurized area — each image was approximately 180 µm × 180 µm). Images were taken before applying a uniaxial strain injury, immediately after injury (0 hour), 1 hour, 2 hours, 4 hours, 9 hours and 24 hours post injury. When images were taken before injury, each position along every microchannel was saved in Slidebook and at every subsequent time point thereafter recalled and reimaged thus allowing for each position along the microchannel/axon to be compared over time.
Ix81 dsu microscope
Manufactured by Olympus
Sourced in United States
The IX81 DSU microscope is a high-performance inverted microscope system designed for advanced imaging applications. It features a digital scanning unit (DSU) and is capable of capturing high-quality, real-time confocal images. The IX81 DSU microscope is a versatile instrument suitable for a wide range of research and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Slidebook software, by Olympus (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Methanol, by Avantor (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using ix81 dsu microscope
1
Longitudinal Axonal Injury Imaging
2
Evaluating RGD-conjugated Fluorescent Dyes for Cell Targeting
3
Immunofluorescent Staining of Cells on Transwell Membranes
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A modification of the standard immunofluorescent staining protocol was used [2 ]. Following overnight growth of HeLa cells, each Transwell membrane apparatus was transferred to a well of a 12-well plate containing PBS/1% FBS, and each membrane was released from the apparatus using a scalpel. The permeable membranes were then washed twice in PBS/1% FBS at room temperature. Cells on membranes were fixed and permeabilized with −20°C methanol (VWR, Radnor, Pennsylvania, USA) for 6 minutes following rehydration with PBS/1% FBS. Table 1 lists antibodies used in this study for staining of cells on membranes. Following antibody staining, membranes were mounted on glass slides using Prolong Gold antifade reagent with DAPI (Invitrogen™ Thermo Fisher Scientific, Waltham, Massachusetts, USA) and covered with coverslips. Control cells were grown on glass coverslips and stained using standard immunofluorescent staining protocols [2 ]. An Olympus IX81-DSU microscope was utilized to visualize the cells, and images were processed using Slidebook 5.0 software (Intelligent Imaging Innovations, Inc., Denver, Colorado, USA).
4
Caveolin-1 Expression in BM-DCs
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Bone marrow cells (2 × 105) were cultured in 12-well plates containing 10 mm coverslips and differentiated into BM-DCs for 6 days using GM-CSF, as described. Cells were stimulated or not with LPS (100 ng/ml) for 24 h, washed three times with cold PBS and then fixed with 4% paraformaldehyde for 10 min at 4°C, washed again, permeabilized using Triton X-100 0.2% in PBS for 10 min, and then blocked with a PBS-BSA 3% solution for 1 h at room temperature. The cells were incubated with anti-CAV1 antibody (1: 200) overnight at 4°C, washed three times with PBS and then incubated 2 h at room temperature in the dark with anti-rabbit second antibody conjugated with Alexa Fluor 488 (Thermo Scientific, Waltham, MA, USA) in a 1: 500 dilution together with 500 nM phalloidin rhodamine (Sigma-Aldrich, St. Louis, MO, USA) and 100 nM 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA). Coverslips were washed and mounted on microscope slides with Mowiol 4-88 (Sigma-Aldrich, St. Louis, MO, USA), and samples were visualized with an Olympus IX81 DSU microscope and analyzed with ImageJ software. Membrane protrusions were counted manually for at least 10 cells per condition in two experiments.
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