After immunohistochemistry or in situ hybridization experiments, images were obtained using a Nikon epifluorescence microscope, a Leica DMI600B fluorescence microscope or a Leica SP5 confocal system.
Dmi600b fluorescence microscope
Manufactured by Leica
The DMI600B is a fluorescence microscope manufactured by Leica. It is designed for high-performance imaging and analysis of fluorescently labeled samples. The microscope features a motorized stage, advanced optics, and a high-sensitivity camera for capturing detailed images.
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4 protocols using dmi600b fluorescence microscope
1
Fluorescence Microscopy Imaging Protocol
2
Macrophage Migration Assay with EPO-Expressing Fibroblasts
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Alveolar macrophages were obtained and cultured using a previously published protocol45 (link). For the migration assay, 4.0 × 104 constitutive EPO-FBs or empty vector-FBs were cultured in 12-well plates. Macrophages were incubated with Vybrant™ DiI membrane dye for 5 min at 37 °C and centrifuged/washed with DMEM media to remove excess dye. 8.0 μm transwells (Falcon #353182) were placed in the 12-well plates containing medium only, EPO-FBs, or empty vector-FBs. In all, 5.0 × 104 macrophages were then placed into the top of the transwell. Quantification of macrophage migration across the transwell membrane was carried out at 90 min and 24 h using a Leica DMI600B fluorescence microscope.
3
Microscopy Imaging Techniques Protocol
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After immunohistochemistry or in situ hybridization experiments, images were obtained using a Zeiss apotome epifluorescence microscope, a Leica DMI600B fluorescence microscope or a Leica SP5 confocal system.
4
Annexin V-Alexa Fluor® 488 & PI Apoptosis Assay
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This experiment was performed using a Dead Cell Apoptosis Kit with Annexin V-Alexa Fluor® 488 & Propidium Iodide (Molecular Probes, USA), following the manufacturer's protocol.
Briefly, treated and untreated trophozoites were washed with chilled PBS and resuspended in 500 µl of 1× binding buffer. Cells were then incubated with Annexin V-Alexa 488 and propidium iodide for 5 min in the dark at room temperature. Data were collected on a BD FACSCalibur instrument controlled by CellQuest Pro software (BD Biosciences, CA, USA) and analyzed with Summit 4.3 (Dako, Fort Collins, CO, USA). A total of 10,000 events were acquired in the regions previously established as those corresponding to G. intestinalis. Alternatively, cells incubated with Annexin V-Alexa 488 and propidium iodide were analyzed by immunofluorescence using a Leica DMI600 B fluorescence microscope.
Briefly, treated and untreated trophozoites were washed with chilled PBS and resuspended in 500 µl of 1× binding buffer. Cells were then incubated with Annexin V-Alexa 488 and propidium iodide for 5 min in the dark at room temperature. Data were collected on a BD FACSCalibur instrument controlled by CellQuest Pro software (BD Biosciences, CA, USA) and analyzed with Summit 4.3 (Dako, Fort Collins, CO, USA). A total of 10,000 events were acquired in the regions previously established as those corresponding to G. intestinalis. Alternatively, cells incubated with Annexin V-Alexa 488 and propidium iodide were analyzed by immunofluorescence using a Leica DMI600 B fluorescence microscope.
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