Apportion of freshly extracted liver tissue was cut into ∼3-mm slices and washed in PBS at RT, fixed in 4% w/v paraformaldehyde for 10 min at RT, and then incubated for 6 h at 4 °C. After a PBS rinse, the samples were dehydrated in 80%, 90%, 95%, and 100% ethanol solutions for 1 h each at 4 °C and finally in dimethyl benzene at RT. Paraffin immersion was at 60 °C for 2 h and then embedding at 4 °C. A rotary microtome was used for cutting 5-μm sections at 4 °C. The sections were then deparaffinized and prepared for H&E staining (Servicebio). Images were acquired under a Leica microscope (Leica DMi8-M).
Dmi8 m
Manufactured by Leica
Sourced in Germany
The DMi8-M is a high-quality microscope system designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research requirements. The core function of the DMi8-M is to provide a versatile and reliable platform for precise microscopic observations and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Merck Group (5 mentions)
Opti mem 1, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Transwell chamber, by Corning (2 mentions)
Gimsa, by Thermo Fisher Scientific (2 mentions)
H e staining, by Wuhan Servicebio Technology (1 mentions)
Giemsa, by Thermo Fisher Scientific (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Giemsa staining solution, by Thermo Fisher Scientific (1 mentions)
Tunel apoptosis detection kit, by Merck Group (1 mentions)
Crystal violet, by Solarbio (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
Opti mem 1 medium, by Thermo Fisher Scientific (1 mentions)
8 μm transwell chamber, by Corning (1 mentions)
Matrigel, by Corning (1 mentions)
Transwell filter, by Corning (1 mentions)
22 protocols using dmi8 m
1
Liver Tissue Preparation for Histology
2
Nuclear Morphology Observation via Hoechst 33342 Staining
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Hoechst 33342 was used to observe nuclear morphological aspects by fluorescence microscopy using a filter from 320 to 350 nm. A7r5 cells were washed twice in phosphate-buffered saline, fixed in phosphate-buffered saline containing 1% (wt/vol) paraformaldehyde (Fisher Scientific, Pittsburgh, PA, USA), rinsed with water, and stained with Hoechst 33342 (final concentration, 5 μg/mL) for 25 min. After staining with Hoechst 33342, the morphological aspects of the nuclei were observed with fluorescence microscopy (Leica DMi8-M, Leica AG, Wetzlar, Germany).
3
Colony Formation Assay Protocol
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Cells in the logarithmic phase were digested with 0.25% trypsin and gently blew into a single cell. The cells were counted and the cell density was adjusted to 1 × 106 cells/mL. Then the cells of each group were reseeded at a density of 50, 100, and 200 cells/mL into 10 cm dish containing 10 mL of preheated medium at 37 °C and incubated for 2 to 3 weeks, the cell culture medium was refreshed every 3 days. It is often observed to terminate the culture when a naked-eye clone appears in the dish. Discard the supernatant and wash 2 carefully with phosphate-buffered saline (PBS). Five milliliters of 4% paraformaldehyde was added to the fixed cells for 15 minutes, then washed with PBS twice, and dyed with proper amount of GIEMSA (Invitrogen) staining solution for 10 to 30 minutes, then slowly washed with running water to remove the staining solution and dried in air. The plate was placed under an inverted microscope (Leica DMI8-M) to observe the number of cell clones and calculate the clone formation rate = the number of formed clones/inoculated cells.
4
Ovarian Cancer Cell Migration and Invasion
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Ovarian cancer cells were resuspended in the Opti-MEMI (Invitrogen, USA) containing 10% BSA (Sigma) with the cell density adjusted to 3 × 104 cells/mL. The migration/invasion experiments were performed by a 24-well plate of 8-μm Transwell chamber (Corning, NY, USA). The cell suspension (100 μL) was added to the upper chamber of each well, while 10% RPMI1640 medium (600 μL) was added to the lower chamber of the well. Then the cells were incubated at 37°C and 5% CO2. In the migration test, the cells were fixed with 4% paraformaldehyde for 30 min after 48 h of incubation, then incubated with 0.2% Triton X-100 (Sigma) solution for 15 min, and dyed with 0.05% gentian violet for 5 min. In the invasion experiment, 50 μL of Matrigel (Sigma) was added to the chamber 48 h before the cells were fixed and stained. The numbers of stained cells were counted under a microscope (DMi8-M, Leica). Five fields of view were randomly selected for counting, and the average number of cells was recorded. The experiment was repeated three times [60 (link)].
5
Neutrophil Migration Assay with CXCL2
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The migration ability of neutrophil to CXCL2 was measured using a Transwell with 3 µm‐pore polyester membrane inserts, with 2 × 105 DiD‐labeled neutrophil seeding in the upper chamber, and 10 ng CXCL2 (20 ng mL−1) added to the lower chamber. After 1 h, fluorescent images of the lower chamber were taken using an inverted fluorescence microscope (Leica, DMi8‐M).
6
Quantifying Cellular Oxidative Stress
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ROS levels were detected using an ROS assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. The CD4+ T cells in each group were incubated with DCFH-DA (10 μM) at 37°C for 20 min and then washed three times. Fluorescence intensity of DCF was measured with a fluorescence microscope (Leica DMi8-M, Germany) at 488 nm (excitation) and 525 nm (emission). The ROS levels were determined according to the fluorescence intensity of DCF.
7
Ovarian Cancer Cell Clonogenic Assay
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Ovarian cancer cells at the logarithmic growth phase were collected, digested by 0.25% trypsin, triturated, counted, and adjusted to 1 × 106 cells/mL. Each group of cells was inoculated with a gradient density of 50, 100, and 200 cells, respectively, in a 10-cm dish that was cultured in a 37 °C 5% CO2 incubator for 2–3 weeks. The culture was terminated when the clone was visible in the culture dish. Then, the cells were fixed by 4% paraformaldehyde (5 mL, Invitrogen) for 15 min and stained by Giemsa staining solution (5 mL, Invitrogen) for 10–30 min. Finally, the cells were washed slowly with PBS and dried in air. The plate was observed under the inverted microscope (DMi8-M, Leica, Wetzlar, Germany). The number of cell clones was recorded and the clone-formation rate was calculated as the number of formed clones / the number of inoculated cells [57 (link), 58 (link)].
8
Colony Formation Assay for PCa Cells
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PCa cells were inoculated into dishes containing 10 mL preheated culture medium, and evenly dispersed by gentle rotation, followed by culture, with the culture medium changed once every 2–3 days. The culture was halted once the clone in the dish was visible to the naked eyes. After removal of supernatant, the cells were washed, and fixed. The fixed cells were stained with an appropriate amount of GIMSA (Invitrogen, USA) for 10–30 min. The number of cell clones was counted under an inverted microscope (Leica DMi8-M, Co. Ltd., Solms, Germany), and the colony formation rate was calculated using the formula: the number of cell clones/the number of inoculated cells × 100%.
9
Immunofluorescence Staining of TLR4 and TIPE2
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RAW264.7 cells on round coverslips and aortic paraffin sections (4 μm) were incubated with 0.3% hydrogen peroxide in methanol for 20 min and heated in EDTA buffer at 150 °C for 30 min to retrieve antigenic activity. Subsequently, cells on coverslips and tissue sections were blocked with goat serum for 30 min, incubated with antibodies specific for TLR4 (1:100) or TIPE2 (1:100) at 4 °C overnight, and then incubated with DyLight™ 594 goat anti-rabbit IgG secondary antibody (1:1,000, A23430, Abbkine, USA) for 2 h at room temperature in dark. Cells were stained with DAPI for 5 min at room temperature. The stained cells and sections were visualized using a fluorescence microscope (DMi8-M, Leica, Germany).
10
TUNEL Assay for Apoptosis Detection
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Apoptotic cells were detected using a TUNEL apoptosis detection kit (Sigma, Concord, CA, USA) according to the manufacturer’s instructions. A7r5 cells were fixed by 4% PFA in PBS for 25 min, incubated in an equilibration buffer for 10 min and then with PBS containing Triton X-100 for 5 min, followed by the TUNEL reaction system at 37 °C for 1 h in the dark. Finally, the cells were stained with Hoechst 33342 for 5 min, and an in situ cell death detection kit was used to detect apoptosis. Images were captured using a fluorescence microscope (Leica DMi8-M).
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