Ix71 inverted fluorescent microscope

Manufactured by Olympus

Sourced in Japan

The IX71 is an inverted fluorescent microscope designed for research applications. It features a high-quality optical system and is capable of capturing detailed fluorescent images. The IX71 is suitable for a variety of cell biology and tissue imaging applications.

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38 protocols using ix71 inverted fluorescent microscope

ROS and Mitochondrial Dysfunction Assay

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ROS generation was measured by fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA). After 12 h exposure to 5 μmol/L RD-1, cells were washed twice with PBS, and then incubated with 10 μmol/L DCFH-DA for 20 min at 37°C. The extracellular DCFH-DA was removed by washing three times with Neurobasal medium free of B27 and then photographed using Olympus IX71 inverted fluorescent microscope. The relative fluorescence intensity was quantified using IPP 6.0 software.
MMP was measured by cationic dye JC-1 which accumulates in mitochondria in a potential-dependent manner. In normal mitochondria, JC-1 polymers produce strong red fluorescence in mitochondria matrix. In unhealthy mitochondria, JC-1 accrues in the cytosol in the form of monomer, producing green fluorescence. After 12 h treatment with 5 μmol/L RD-1, cells were washed twice with PBS, and then incubated with 10 mg/mL JC-1 for 15-20 min at 37°C. Then the cells were washed three times before they were suspended in incubation buffer. The images were captured using Olympus IX71 inverted fluorescent microscope and counted blindly. Mitochondrial depolarization degree was expressed as the ratio of red/green neurons numbers.

Wang M., Feng L., Zheng J., Liu J., Fan S., Zhao J., Yang N., Liu Y., Yang Z., Ye C, & Zuo P. (2017). Improvement of mitochondrial function mediated the neuroprotective effect of 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone in rats with cerebral ischemia-reperfusion injuries. Oncotarget, 8(37), 61193-61202.

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Detecting Cell Death Mechanisms

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To examine the status of cell death, we used AO/EB mixture (each 100 μg/mL in PBS). iPSCs and iPSC-Diff cultured on 12-well culture plates were treated with ECR or doxorubicin for 24 h and then treated AO/EB mixture for 20 min at RT. After washing with mTeSR1 medium, cells were observed under an Olympus IX71 inverted fluorescent microscope. Viable, early apoptotic, late apoptotic, and necrotic cells were identified as green, condensed green, yellow to orange, and red, respectively. To detect apoptotic nuclei, cells grown on the confocal dishes were treated with ECR or doxorubicin for 24 h, fixed with 10% formalin for 30 min at RT, stained with DAPI solution (1 μg/mL) for 10 min at RT, and then observed under an Olympus IX71 inverted fluorescent microscope.

Kim A., Baek S.J., Shin S., Lee S.Y, & Chung S.K. (2023). An Ethanol Extract of Coptidis rhizoma Induces Apoptotic Cell Death in Induced Pluripotent Stem Cells and Suppresses Teratoma Formation. Nutrients, 15(10), 2364.

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Immunofluorescence Assay for Macrophage Infiltration

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An immunofluorescence assay to detect macrophage infiltration in mouse colonic mucosa was performed according to a previously described method (21 (link)). Briefly, after colons were excised and rinsed with PBS, colon tissues were fixed in 10% PBS-buffered formalin at room temperature for 24 h, then embedded in paraffin, cut into 5 µm sections and mounted on glass slides. Sections were subsequently deparaffinized in xylene and rehydrated in a graded series of ethanol solutions (50 75, 85, 95 and 100%, 3 min/solution). Sections were then blocked with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) in Tris-buffered saline (TBS) for 90 min at room temperature, then immunostained overnight at 4°C with anti-CD68 antibody (1:100) and 5% BSA in TBS. Following immunostaining, sections were washed three times with TBS, then incubated with Alexa Fluor 488-conjugated secondary antibody (1:200; cat. no. 715-547-003; Jackson Immunoresearch Laboratories, USA) in TBS for 2 h at room temperature in the dark. Sections were then mounted with mounting medium containing 5 mg/ml DAPI (Beyotime Institute of Biotechnology, Shanghai, China) for nuclear counterstaining and visualized under an Olympus IX71 inverted fluorescent microscope (Olympus Corporation, Tokyo, Japan).

Zhang L.C., Wang Y., Tong L.C., Sun S., Liu W.Y., Zhang S., Wang R.M., Wang Z.B, & Li L. (2017). Berberine alleviates dextran sodium sulfate-induced colitis by improving intestinal barrier function and reducing inflammation and oxidative stress. Experimental and Therapeutic Medicine, 13(6), 3374-3382.

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Paraffin-embedded Tissue Histology Protocol

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Paraffin-embedded tissues were cut into 3 μm sections. H&E staining was performed according to the standard protocol. Briefly, sections were treated as follows: dewaxing (xylene, 2 × 2 min), rehydration (90% ethanol for 10 min, 80% ethanol for 10 min, and 75% ethanol for 10 min), hematoxylin staining (5 min, G1004, Servicebio, Wuhan, Hubei, China), differentiation (2 s, G1039, Servicebio), bluing (2 s, G1040, Servicebio), dehydration (85% ethanol for 5 min and 95% ethanol for 5 min), eosin staining (5 min, G1001, Servicebio), dehydration (anhydrous ethanol for 3 × 5 min), and hyalinization (xylene, 2 × 5 min). Sections were then sealed with neutral gum (10004160, Sinopharm, Beijing, China) and photographed using an Olympus IX-71 inverted fluorescent microscope (Olympus, Tokyo, Japan).

Shu Y., Peng F., Zhao B., Liu C., Li Q., Li H., Wang Y., Jiang Y., Lu T., Wang Q., Sun J., Feng H., Lu Z., Liu X., Wang J, & Qiu W. (2023). Transfer of patient’s peripheral blood mononuclear cells (PBMCs) disrupts blood–brain barrier and induces anti-NMDAR encephalitis: a study of novel humanized PBMC mouse model. Journal of Neuroinflammation, 20, 164.

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Live Cell Imaging of Fluorescent Proteins

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As previously described [61 (link)], imaging was carried out with a DeltaVision Elite (Applied Precision, Buckinghamshire, UK) comprising an Olympus IX71 inverted fluorescent microscope, and Olympus UPlanSApo ×100, NA 1.40, oil immersion objective and a CoolSNAP HQ2 camera cooled to −30 °C (Roper Scientific, Sarasota, FL, USA). Cells were adhered to 35 mm glass culture dishes (MatTek, Ashland, MA, USA) precoated with 0.2 mg ml⁻¹ soybean lectin (Calbiochem, Merck Millipore) and immersed in EMM-N media. Culture dishes were placed on the inverted microscope stage in an Environmental Chamber at 28 °C.
For live cell imaging, mCherry, YFP (or GFP) and Cerulean signals were captured with 1.2 s (32% filter), 1.5 s (32% filter) and 0.5 s (32% filter) exposures using Optical Axis Integration, which acquires 3.6 μm of z-axis by a continuous z sweep. This was repeated every 300 s for approximately 12 h. Images were deconvolved and analysed using SoftWoRx 5.5 (Applied Precision). Dead cells observed during the imaging and the subjects moving out of focus were excluded from the study.

Moiseeva V., Amelina H., Collopy L.C., Armstrong C.A., Pearson S.R, & Tomita K. (2017). The telomere bouquet facilitates meiotic prophase progression and exit in fission yeast. Cell Discovery, 3, 17041-.

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Mitochondrial Membrane Potential Assay

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iPSCs grown on confocal dishes were treated with ECR or doxorubicin for 12 h or 3 h, respectively. After washing with mTeSR1 medium, cells were incubated with JC-1 (5 μg/mL, Invitrogen/Molecular probes) in the dark for 10 min at 37 °C. Cells were washed with mTeSR1 medium and then observed under an Olympus IX71 inverted fluorescent microscope.

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mCherry Fluorescence Detection on EMVs

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For detection of mCherry fluorescence on EMVs derived from mCherry-labeled, 143B luciferase–expressing, puromycin-resistant cells, EMV suspensions were examined microscopically using the Olympus (Center Valley, PA) IX71 inverted fluorescent microscope equipped with a xenon arc lamp and monochromatic complementary metal oxide semiconductor camera. In addition, flow cytometric data were acquired on EMV suspensions using the BD LSR II flow cytometer integrated with FACSDiva software (BD Biosciences, San Jose, CA, USA).

Garimella R., Washington L., Isaacson J., Vallejo J., Spence M., Tawfik O., Rowe P., Brotto M, & Perez R. (2014). Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment. Translational Oncology, 7(3), 331-340.

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Apoptosis Detection in HUVECs using Hoechst 33342

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As previous described [6 (link)], we used Hoechst 33342 staining for detecting apoptotic HUVECs. Briefly, HUVECs were plated in 24-well plate at density of 1 × 10⁴ cells/well. After being transfected with miRNA mimics and plasmids with indicated time, cells were stained with Hoechst 33342 for 10 min at 37°C. Then the fluorescent images were captured by Olympus IX71 Inverted Fluorescent Microscope (Olympus, Japan). The apoptotic cell number was quantified with Image-Pro Plus 5.1 Image Analysis Software.

Zhao X., Yi Y., Meng C, & Fang N. (2019). MiRNA-575 suppresses angiogenesis by targeting Rab5-MEK-ERK pathway in endothelial cells. Bioscience Reports, 39(1), BSR20181218.

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Immunofluorescence Staining Protocol for Pluripotent Stem Cells

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Cells were fixed in 4% PFA (Sigma), permeabilized with 0.5% Triton X-100 (Sigma) in DPBS (ThermoFisher), and blocked with 5% goat serum (ThermoFisher). All antibodies used in this study are detailed in Supplementary Table 9 (for example, primary antibodies used were rabbit anti-NANOG polyclonal (1:100, Abcam) and mouse anti-TRA-1-60 IgM (1:300, BD Biosciences)). Primary antibody incubation was conducted overnight at 4 °C on shakers followed by incubation with secondary antibodies (1:400) for 1 h. After labelling, cells were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1:1,000, ThermoFisher) for 30 min. Images were taken using an IX71 inverted fluorescent microscope (Olympus). The following markers were assessed for respective differentiation assays: SOX17 and FOXA2 for endoderm progenitor differentiation experiments; SOX1 and PAX6 for neural differentiation experiments; PAX3 and PAX7 for skeletal muscle differentiation experiments; GATA6 and TTF1 for lung differentiation experiments.

Buckberry S., Liu X., Poppe D., Tan J.P., Sun G., Chen J., Nguyen T.V., de Mendoza A., Pflueger J., Frazer T., Vargas-Landín D.B., Paynter J.M., Smits N., Liu N., Ouyang J.F., Rossello F.J., Chy H.S., Rackham O.J., Laslett A.L., Breen J., Faulkner G.J., Nefzger C.M., Polo J.M, & Lister R. (2023). Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature, 620(7975), 863-872.

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Immunofluorescence Staining of Cells and Tissue Sections

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Cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature, after being washed with PBS three times, the cells were blocked and permeated with PBS containing 5% BSA and 0.3% Triton for 1 hr at room temperature. Then cells were incubated with the relevant primary antibody at 4°C overnight. After a thorough washing, cells were incubated with the appropriate fluorescence-conjugated secondary antibody for 1 hr at room temperature. Nuclei were stained with Hoechst 33,342 (10 μg/mL).
For staining of frozen tissue sections, fresh liver specimens were embedded in cryo-embedding medium and stored in frozen blocks at −80°C prior to sectioning. Sections of 10 μm thickness were fixed with 4% PFA for 30 min, and then blocked with 5% BSA. The sections were then incubated with the correct primary antibodies and secondary antibodies similar to cell staining. Nuclei were stained with Hoechst 33,342 (10 μg/mL). Images were captured with an Olympus IX71 inverted fluorescent microscope. Antibodies used in this study are as follows: anti-E-cadherin (CST, 24E10), anti-albumin (Bethyl Laboratories, A90-234A), anti-ZO-1 (Gibco, 40–2200), anti-Cyp1a2 (Abcam, ab22717), anti-Cyp2c9 (Abcam, ab4236), anti-Cyp2c19 (Abcam, ab137015), and anti-Fah (Abcam, ab81087).

Guo R., Tang W., Yuan Q., Hui L., Wang X, & Xie X. (2017). Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor. Stem Cell Reports, 9(2), 499-512.

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