For DAPI pollen in 0 ~ 2 mm, 2 ~ 4 mm, 4 ~ 6 mm, 6 ~ 8 mm, and 8 ~ 10 mm TO1000 oral buds were put on microscope slides and stained with several drops of DAPI-staining solution, as described [66] . The pollen nuclei were inspected under an Olympus BX51 uorescence microscope (Tokyo, Japan) with a DAPI lter.
Bx51 uorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The BX51 is a fluorescence microscope designed for biological and life science research. It features a high-intensity illumination system and advanced optics to enable clear visualization and imaging of fluorescently-labeled samples. The BX51 supports a range of objective lenses and filter sets to accommodate various fluorescent dyes and applications.
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Lab products found in correlation
Bcl 2, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Novus Biologicals (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
α actinin, by Merck Group (1 mentions)
Tmr red, by Roche (1 mentions)
Sigmaplot 12, by Grafiti LLC (1 mentions)
Apoptag fluorescein in situ apoptosis detection kit, by Merck Group (1 mentions)
6 protocols using bx51 uorescence microscope
1
DAPI Staining of Pollen Nuclei
2
Immunofluorescence Analysis of Bax and Bcl-2
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To determine Bax and Bcl-2 expression pro les, an immuno uorescence staining was used as previously described (Onder et al. 2022 (link)). Cells were incubated on coverslips in a 24-well plate. After cells were treated with HES for 24 hours, they were xed in 10% formaldehyde. Then, the procedure of immuno uorescence staining was applied. Cells were treated with Bax (1:100, primary antibody, Novus Biologicals, Littleton, USA) and then Bcl-2 (1:30, primary antibody, Thermo, Rockford, USA). For each group, 400x images were obtained from ten separate microscopic elds using an Olympus BX51 uorescence microscope (Olympus, Tokyo, Japan). The intensity of Bax and Bcl-2 primary antibodies' immunoreactivity was measured using Image J software (Bethesda, USA).
3
Immunofluorescent Staining of Myocardial Tissue
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Myocardial tissue was xed with 10% buffered formalin and embedded in para n, after which it was cut into 4µm thick slices. The sections were incubated in 0.1% (vv - 1 ) Triton X-100 for 8min and blocked with normal horse serum for 30min at room temperature. The sections were then incubated with cleaved-caspase3 antibody (1:600, 9664, Cell Signaling, Inc. Danvers, MA, USA) and α-actinin (1:600, A7811, Sigma-Aldrich, St Louis, MO, USA) at 4°C overnight,[26] and then with corresponding uorescent secondary antibody for 1.5h at room temperature in dark. The sections were then stained with DAPI and observed by Olympus BX51 uorescence microscope (Olympus America Inc., Center Valley, PA, USA).
ImageJ software (NIH) was used to calculate the positive area.
ImageJ software (NIH) was used to calculate the positive area.
4
TUNEL Staining for Apoptosis Analysis
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TUNEL staining was performed in accordance with the instructions from TMR Red (Roche, Mannheim, Germany). [27] Tissues were para n-embedded and cut into 4µm thick slices. The myocardial cells were washed twice with PBS and xed for 15min at room temperature. The slices or cells were incubated in 0.1% (VV-1) Triton X-100 for 8min and sealed with 3% (WV-1) bovine serum for 1h at room temperature.
Each section was incubated with TUNEL conjugated dUTP at 37°C for 60min. After staining with α-actin and 4 ', 6-diamino-2-phenylindoles (DAPI), sections were stored in a refrigerator at -20°C. Images were obtained using an Olympus BX51 uorescence microscope (Olympus America Inc., Center Valley, PA, USA) at 20× magni cation. Image J (NIH) was used to calculate the number of TUNEL-positive cells and the total cells in each eld.
Each section was incubated with TUNEL conjugated dUTP at 37°C for 60min. After staining with α-actin and 4 ', 6-diamino-2-phenylindoles (DAPI), sections were stored in a refrigerator at -20°C. Images were obtained using an Olympus BX51 uorescence microscope (Olympus America Inc., Center Valley, PA, USA) at 20× magni cation. Image J (NIH) was used to calculate the number of TUNEL-positive cells and the total cells in each eld.
5
Detecting ROS in T. vaginalis-infected SiHa cells
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SiHa cells (1 × 10 5 cells/well; 12-well plate) seeded on a coverslip were cultured in DMEM supplemented with 10% FBS and the culture medium was replaced when the cells reached 80% con uence. To evaluate the generation of ROS, SiHa cells were infected with live T. vaginalis (MOI 2 and 5) for 2 h, and 6h, and then incubated with 5 𝜇M MitoSOX reagent or CellROX reagent and then incubated for 10 min at 37°C, 5% CO 2 in the dark. The stained cells were imaged using an Olympus BX51 uorescence microscope. All experiments were performed on triplicate samples and uorescence intensity was calculated using ImageJ software, and graph was plotted using SigmaPlot 12.5 (Systat Software, San Jose, CA).
6
Quantifying HES-Induced Apoptosis
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To assess HES-mediated apoptotic cell death, the ApopTag Fluorescein In Situ Apoptosis Detection Kit (EMD Millipore, Darmstadt, Germany) was as previously described (Baran et al. 2020 (link)). An Olympus BX51 uorescence microscope (Olympus, Tokyo, Japan) was used to observe the apoptotic cells (magni cation, x400). A total of 10 elds of view were randomly selected for analysis.
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