Samples were fixed with 4% PFA/PBS, permeabilised with 0.05% Triton/PBS and blocked with 5% goat serum/PBS for 60 min. Samples were incubated overnight at 4 °C on a rocker with primary antibody mouse-α-MYOGENIN 1:10 (DSHB, F5D). Next day, samples were washed in PBS for 5 min three times and incubated for 60 min at room temperature with α-mouse 488 1:500 (ThermoFisher, A-11001) diluted in PBS. After further washes, nuclei were counterstained with DAPI 1:1000 and mounted in vectashield (VWR, 101098–042). Samples were viewed and imaged on a Leica AxioVert 200 M.
Axio vert
Manufactured by Leica
Sourced in Germany
The Axio Vert is a microscope designed for routine laboratory work. It features a sturdy, ergonomic design and offers high-quality optics for precise observation and documentation. The Axio Vert is suitable for a variety of applications in materials science, life sciences, and other research fields.
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Lab products found in correlation
5 protocols using axio vert
1
Immunofluorescence Analysis of Myogenin
2
Quantifying Cell Apoptosis by Hoechst Staining
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Cell apoptosis was determined by Hoechst 33258 staining. Brie y, cells were seeded into 24-well plates at a density of 1×10 5 cells/well for 24 h. The cells were challenged with TNF-α or Vehicle for 24 h and then washed with PBS. All cells were xed using paraformaldehyde for 20 min at room temperature followed by washing twice with PBS. Afterwards, cells were stained with Hoechst 33258 solution (10 μmol/L) for 5 min at room temperature. After being washed twice with PBS, the images of the cells were visualized by a uorescence-capable microscope (Leica, Axio Vert, Germany). The percentage of apoptotic cells was calculated by counting the total number of cells and the number of Hoechst-stained cell.
3
Cell-in-Cell Structures Microscopic Analysis
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Cells were fixed in 2.5% glutaraldehyde with 2% formalin in 0.1 M PBS buffer (pH 7.4) for 1 h. DNA was stained with DAPI for 10 min. After phase-contrast and fluorescent images of cell-in-cell structures were taken (by Axiovert 200 M, × 63 and × 20), the selected areas of interest were marked using a permanent marker on the cover slips and identified under the Differential Interference Contrast (DIC) microscope (Leica, objectives Plan × 10/0.22 and × 20/0.30). The cell layer around the marked areas was then brushed off under the DIC microscope using a fine needle. The samples were carefully washed to remove oil and scraped cells and subjected to a standard SEM procedure as described above.
4
Confocal Microscopy Protocol for Colocalization
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Images were acquired with Zeiss LSM510 (Zeiss) and Leica SPE (Leica Microsystems) confocal units attached to Zeiss Axiovert and Leica IM8 microscopes, respectively. Colocalization was performed using z-stacks. Proliferation assays used a 20Â air objective, and all other experiments were imaged using a 40Â oil pH2 objective. Zeiss image manipulation software (ZEN) was used for editing.
5
Quantifying Cell Apoptosis by Hoechst Staining
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Cell apoptosis was determined by Hoechst 33258 staining. Brie y, cells were seeded into 24-well plates at a density of 1×10 5 cells/well for 24 h. The cells were challenged with TNF-α or Vehicle for 24 h and then washed with PBS. All cells were xed using paraformaldehyde for 20 min at room temperature followed by washing twice with PBS. Afterwards, cells were stained with Hoechst 33258 solution (10 μmol/L) for 5 min at room temperature. After being washed twice with PBS, the images of the cells were visualized by a uorescence-capable microscope (Leica, Axio Vert, Germany). The percentage of apoptotic cells was calculated by counting the total number of cells and the number of Hoechst-stained cell.
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