To measure the MMP, P7C3- or MPP+-treated MES23.5 cells were incubated with 100 nmol/l TMRM (Sigma) in PBS for 15 min at 37 °C. Healthy mitochondria were indicated with TMRM in red, while damaged mitochondria rapidly lost the red fluorescence, implying the loss of MMP. After incubation, MES23.5 cells were immediately washed with PBS and then resuspended in PBS. Finally, the cells were observed using an inverted IX71 microscope system (Olympus).
Ix71 microscope system
Manufactured by Olympus
Sourced in Japan, United States
The Olympus IX71 microscope system is a high-performance inverted microscope designed for a wide range of applications. It features a sturdy, ergonomic design and advanced optics to deliver clear, detailed images. The IX71 supports various imaging techniques, including brightfield, darkfield, and phase contrast.
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Lab products found in correlation
Anti th antibody, by Merck Group (2 mentions)
Anti iba1 antibody, by Fujifilm (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Rhodamine red or fitc green conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
F view soft imaging system camera, by Olympus (1 mentions)
Vectashield reagent, by Vector Laboratories (1 mentions)
Af6000, by Leica (1 mentions)
Blocking one reagent, by Nacalai Tesque (1 mentions)
Hochest 33342, by Merck Group (1 mentions)
Propidium iodide, by Beyotime (1 mentions)
Ab97040, by Abcam (1 mentions)
Ab242197, by Abcam (1 mentions)
11 protocols using ix71 microscope system
1
Measuring Mitochondrial Membrane Potential
2
Quantifying Neuroinflammation in Parkinson's Model
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After the treatments of Ramelteon and LPS or MPTP described above, the mice were perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The mice brains were then removed and post‐fixed in the same fixation agent overnight at 4°C, followed by a treatment with 30% sucrose at 4°C with another night. Serial 20 μM‐thick mouse midbrain slices were cut with a freezing microtome. Immunohistochemical staining was conducted with anti‐MT1 antibody, anti‐Iba1antibody (Wako Chemicals), anti‐GFAP, and anti‐TH antibodies (Millipore) against six slices per mouse (120 μm interval). After incubation with primary antibodies at room temperature for 6 hr, the slices were incubated with rhodamine (red)‐or FITC (green)‐conjugated secondary antibody (Invitrogen) for 2 hr. Thereafter, the slices were stained with DAPI for 5 min and observed using an inverted IX71 microscope system (Olympus). The number of TH+ neurons and the fluorescence intensity of MT1, Iba1, and GFAP were counted using Image J (National Institute of Health).
3
Immunohistochemical Quantification of Dopaminergic Neurons
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Male C57BL/6 mice, 25–30 g, were administrated P7C3 or MPTP as described in the animal experiments above. Then, the mice were perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). After perfusion, the mice brains were harvested for post-fixation in the same fixing agent overnight at 4 °C, followed by the treatment with the 30% sucrose at 4 °C for another night. Serial 20 μM-thick mouse midbrain slices were prepared with a freezing microtome. Immunohistochemical staining was conducted using anti-TH antibodies (Millipore) against six slices per mouse (120 μm interval). After incubation with primary antibody at room temperature for 6 h, the slices were incubated with rhodamine (red)- or FITC (green)-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 2 h. Finally, the slices were stained with DAPI for 5 min and observed using an inverted IX71 microscope system (Olympus). TH+ neurons were counted with Image J software (National Institutes of Health, Bethesda, MD, USA) for each slice by two blinded investigators.
4
Fluorescence Microscopy Using Olympus IX71
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The fluorescence microscopy was performed using Olympus IX71 microscope system with Olympus LUCPlanFLN 40x objective, with attached Olympus F-View Soft Imaging System camera using the CellSens acquisition software version 1.3 (all Olympus Corporation, Tokyo, Japan).
5
Tracking TNBC Cell Intravasation
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TNBC intravasation from the tissue compartment, across the EC barrier, into the vascular channels was monitored by fluorescence imaging. For this purpose, CellTracker CM-DiI dye, which is non-toxic, well retained, and allows for multigenerational tracking of cellular movements (Krishnamurthy et al., 2008 (link)), was used. Briefly, TNBC cells were stained with CM-DiI dye before they were introduced into the tissue compartment. Meanwhile, HUVECs were stained with CellTracker Green CMFDA dye for better visualization of TNBC intravasation. Fluorescence images were acquired using the Olympus IX71 microscope system, as explained above. Images were then analyzed in cellSens Dimension software for quantifying intravasation. This was achieved using the “Count and Measure” function in cellSens Dimension software, where ROIs were drawn in different areas of the image as shown in Supplementary Figure S3 . The obtained cell numbers were then analyzed and quantified (Detailed steps are given in Supplementary Material ).
6
Measuring Mitochondrial Membrane Potential
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To measure ΔΨm, HepG2 cells were treated with 100 nmol/L TMRM (Thermo Fisher) for 15 min at 37°C after treatment with sorafenib or regorafenib. Healthy cells with ΔΨm are labeled with TMRM and present red fluorescence, while damaged cells with decreased ΔΨm exhibit decreased TMRM labeling (Yu et al., 2016 (link)). After incubation, the HepG2 cells were washed with PBS. Finally, the cells were imaged with an inverted IX71 microscope system (Olympus, Tokyo, Japan).
7
Immunofluorescence Microscopy Protocol
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Cells on dishes or wells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and permeabilized using PBS containing 0.1% Tween-20. Permeabilized cells were blocked with Blocking One reagent (Nacalai Tesque); subsequently they were incubated with primary antibodies and then with fluorescence-labeled secondary antibodies. Thereafter the cells were mounted using Vectashield reagent (Vector Laboratories, Burlingame, CA) for microscopic observation. Fluorescence images were captured using a DMI4000 microscope system (Leica, Wetzlar, Germany) and analyzed using AF6000 software (software attached to DMI4000; Leica) or captured using an IX71 microscope system (Olympus, Tokyo, Japan) and analyzed using IX2-BSW software (software attached to IX71; Olympus).
8
Microglia-mediated Neuronal Apoptosis Assay
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BV2 cells were transfected with Atg5 siRNA or control siRNA for 48 h as mentioned above. For rapamycin group, cells were pretreated with 0.2 μg/ml rapamycin for 0.5 h. Cells were then treated with TNF-α for 24 h, and replaced by culture in fresh medium for another 24 h to produce the microglia conditioned medium (CM). After that, the microglia CM was transferred into MES23.5 cells and cultured for 24 h. Thereafter, cells were incubated with Hochest 33342 (Sigma) and propidium iodide (PI, Beyotime, Shanghai, China) for 5 min, and then washed with PBS. Images were taken using an inverted IX71 microscope system (Olympus, Tokyo, Japan). The hochest and PI stained cells were counted manually, and at least 500 cells per group were counted.
9
Retinal Layers Thickness Measurement
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The thickness of the retina layers 300 μm adjacent to the optic nerve was measured in paraffin embedded retinal sections stained with hematoxylin and eosin (H&E). The microphotographs were taken under 20× objective of an Olympus IX71 microscope system (Olympus, Japan) linked with a digital camera (SPOT CCD Digital Camera, Diagnostic Instruments Inc., Sterling Heights, MI, USA). The retinal layers being assessed were as follows: (a) outer lim- Cells in the GCL with pyknotic nuclei were defined as dead cells. The number of the dead cells in the GCL layer was counted in only one of the four consecutive sections of one slide to avoid double counting. The differences between contralateral and ipsilateral sides in Epac-wildtype and Epac-deficient mice were compared.
10
Immunohistochemical Analysis of RelB
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RelB expression was analyzed by immunohistochemistry (IHC) in four micrometer‐thick, formalin‐fixed, paraffin‐embedded (FFPE) sections. Tissue IHC was performed using a standard peroxidase‐based staining method. Tissue sections were incubated in a dry oven at 60°C for one hour, dewaxed in xylene for 3 × 10 minutes, and rehydrated with graded ethanol in 100%, 100%, 95%, 90%, 80%, and 70% ethanol for five minutes each. Antigen retrieval was then performed by pretreatment of the slides in 0.01 M citrate buffer (pH 6.0) using a microwave oven. Subsequently, the sections were treated with 3% hydrogen peroxide (H2O2) for 10 minutes in order to block endogenous peroxidase. The sections were washed with 1 × phosphate buffered saline (PBS; pH 7.4) and were incubated with rabbit anti‐RelB antibody (dilution 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. The sections were then washed with 1 × PBS and incubated with biotinylated goat anti‐rabbit immunoglobulin G. For each sample, the omission of primary antibody was used as a negative control. Finally, 3, 3‐diaminobenzine (DAB) was used to visualize the immunoreactive products. The results were evaluated by System Microscope IX71 (Olympus America Inc., Center Valley, PA, USA).
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