Epifluorescence microscope

Manufactured by Olympus

Sourced in Japan, Germany, United States, Panama

The Epifluorescence microscope is an optical imaging tool designed to visualize fluorescently labeled samples. It utilizes a high-intensity light source and specialized filters to excite fluorophores within the sample, allowing for the observation of specific cellular structures or molecules of interest. The core function of the epifluorescence microscope is to provide a non-invasive method for studying the distribution and dynamics of fluorescently tagged targets within a sample.

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Lab products found in correlation

In situ cell death detection kit, by Roche (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions) 35 mm glass coated wells, by Iwaki (4 mentions) Alexa fluor 488 green conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions) Alexa fluor 594 red conjugated anti mouse igg, by Thermo Fisher Scientific (3 mentions) Cell a imaging software, by Olympus (3 mentions) Vectashield, by Vector Laboratories (3 mentions) Anti fade mounting solution, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Cam xm10 charge coupled device ccd, by Olympus (2 mentions) Ammonium isothiocyanate, by Merck Group (2 mentions) Biotinylated cd1a clone hi149, by BioLegend (2 mentions) Strepavidin alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Rabbit anti ki67, by Abcam (2 mentions) Saponin, by Merck Group (2 mentions) Protein block solution, by Agilent Technologies (2 mentions) Ab16669, by Abcam (2 mentions) Mca48r, by Bio-Rad (2 mentions) Fluoromount g, by Southern Biotech (2 mentions)

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122 protocols using epifluorescence microscope

TUNEL Assay for Apoptosis Detection

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For TUNEL assays, we used the DeadEnd Colorimetric TUNEL Assay kit (Promega, Madison, WI) performed according to the manufacturer’s instructions. Briefly, paraffin-embedded slides were deparaffinized and rehydrated. Pre-equilibrated slides were labeled with a labeling DNA-strand break solution containing a biotinylated nucleotide mix (60 min at 37°C). After several washes in two times SSC and PBS, slides were blocked with hydrogen peroxide (3–5 min at room temperature). After several washes in PBS, the slides were mounted with mounting solution with DAPI. Apoptotic cells on the slides were observed under an Olympus epifluorescence microscope (X10 magnification; Olympus, Center Valley, PA) in randomly chosen fields. For detection of apoptosis in a time-dependent manner in vitro, PC-3 cells were grown in microscopic slide culture chambers (BD Bioscience, San Jose, CA) and cells were treated with Ad.5/3-CTV and BI-97C1 (Sabutoclax, Sanford Burnham Medical Research Institute, La Jolla, CA) after which the cells were fixed with 4% formaldehyde at the indicated time and TUNEL assays were performed as per the manufacturer’s instruction using an Olympus epifluorescence microscope (X10 magnification; Olympus, Center Valley, PA) (Dash et al., 2011a (link)).

AZAB B.M., DASH R., DAS S.K., BHUTIA S.K., SARKAR S., SHEN X.N., QUINN B.A., DENT P., DMITRIEV I.P., WANG X.Y., CURIEL D.T., PELLECCHIA M., REED J.C., SARKAR D, & FISHER P.B. (2014). Enhanced Prostate Cancer Gene Transfer and Therapy Using a Novel Serotype Chimera Cancer Terminator Virus (Ad.5/3-CTV). Journal of cellular physiology, 229(1), 34-43.

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Immunocytochemistry of Tricellulin and Occludin

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For immunocytochemistry, cells were grown on 35-mm glass-base dishes (Iwaki, Chiba, Japan) coated with rat tail collagen. They were fixed with an ethanol and acetone 1:1 mixture at −20 °C for 10 min. Staining was performed as described previously22 (link) using the primary polyclonal anti-tricellulin antibody and monoclonal anti-occludin antibody. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan) and a confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany).

Takasawa A., Murata M., Takasawa K., Ono Y., Osanai M., Tanaka S., Nojima M., Kono T., Hirata K., Kojima T, & Sawada N. (2016). Nuclear localization of tricellulin promotes the oncogenic property of pancreatic cancer. Scientific Reports, 6, 33582.

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Immunofluorescence Analysis of Cytoskeletal Proteins

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Cells cultured in 35-mm glass-coated wells (Iwaki, Chiba, Japan) were fixed with cold acetone and ethanol (1:1) at −20 °C for 10 min. After rinsing in PBS, the cells were incubated with anti-LSR (1:100), anti-pYAP (1:100), anti-AMOT (1:100), and anti-Merlin (1:100) antibodies at room temperature for 1 h. Alexa Fluor 488 (green)-conjugated anti-rabbit IgG and Alexa Fluor 594 (red)-conjugated anti-mouse IgG (Invitrogen) were used as secondary antibodies. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan) and a confocal laser scanning microscope (LSM5; Carl Zeiss, Jena, Germany).

Shimada H., Abe S., Kohno T., Satohisa S., Konno T., Takahashi S., Hatakeyama T., Arimoto C., Kakuki T., Kaneko Y., Takano K.I., Saito T, & Kojima T. (2017). Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer. Scientific Reports, 7, 37049.

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Immunofluorescence Imaging of Tight Junction Proteins

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The cultured cells in 35-mm glass-coated wells (Iwaki, Chiba, Japan) were fixed with cold acetone and ethanol (1:1) at −20°C for 10 min. After rinsing in PBS, the cells were incubated with anti-cytokeratin 7 (1:200), anti-LSR (1:100), anti-tricellulin (1:100), and anti-vimentin (1:100) antibodies at room temperature for 1 h. Alexa Fluor 488 (green)-conjugated anti-rabbit IgG and Alexa Fluor 594 (red)-conjugated anti-mouse IgG (Invitrogen) were used as secondary antibodies. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan) and a confocal laser scanning microscope (LSM5; Carl Zeiss, Jena, Germany). Some images were captured using a Zeiss Elyra PS1 SIM equipped with a Zeiss Plan Apochromat inverted 63x/1.40 oil immersion objective lens using an Andor EM-CCD iXon 885 camera and a 1.6 x tube lens at room temperature (Carl Zeiss, Jena, Germany).

Shimada H., Satohisa S., Kohno T., Takahashi S., Hatakeyama T., Konno T., Tsujiwaki M., Saito T, & Kojima T. (2016). The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells. Oncotarget, 7(19), 27735-27752.

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Visualizing 4-Hydroxy-2-Nonenal in HT1080 Cells

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HT1080 cells were cultured on Lab‐Tek chamber glass slides at 37°C for 48 h. Indirect immunofluorescence staining was performed as described previously.22 Cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton‐X‐100 and then incubated with primary antibodies followed by incubation with Alexa 488‐conjugated, species‐specific anti‐IgG secondary antibodies (Molecular Probes, OR, USA). Anti‐4‐hydroxy‐2‐nonenal mouse antibody (clone HNEJ‐2, JalCA, Shizuoka, Japan) was diluted before use (1:25). Intrinsic mRFP and antibody staining signals were observed with an FV10i confocal microscope (Olympus, Tokyo, Japan) or with an epifluorescence microscope (Olympus BX‐50) equipped with a SenSys charge‐coupled device camera (Photometrics). Fluorescence images were collected by a single rapid scan with identical parameters (e.g. contrast and brightness) for all samples.

Shintoku R., Takigawa Y., Yamada K., Kubota C., Yoshimoto Y., Takeuchi T., Koshiishi I, & Torii S. (2017). Lipoxygenase‐mediated generation of lipid peroxides enhances ferroptosis induced by erastin and RSL3. Cancer Science, 108(11), 2187-2194.

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Embryo Quality Assessment via Microscopy

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All cleaved embryos were morphologically observed under an inverted microscope (40 × magnification) to evaluate morulae and blastocysts on day 5 and day 7, respectively (day of IVF = day 0). On day 7, all embryos were fixed in 4%
(w/v) paraformaldehyde and stained with 0.1 µg/ml DAPI. Total cell counts were performed using an epifluorescence microscope (Olympus, Japan). The percentages of morulae (> 16 cells without blastocoels) and blastocysts (> 50
cells with blastocoel formation) were recorded.

ARAYATHAM S., TIPTANAVATTANA N, & THARASANIT T. (2017). Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes. The Journal of Reproduction and Development, 63(5), 511-517.

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Assessing Neuro2a Cell Viability after Transfection

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To determine Neuro2a cell viability after transfection with α7 nAChR, the chaperone NACHO and a calcium sensor Case12-coding plasmids, cells were incubated with 20 nM tetramethylrhodamine ethyl ester (TMRE, Invitrogen, USA) for 20 min and then washed with the buffer containing 140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4. Identification of the non-viable cells was performed by staining with propidium iodide (50 ng/ml, BD Biosciences, USA) for 5 min followed by brief washing with the buffer. The bright field and fluorescent pictures were taken with an epifluorescence microscope (Olympus, Japan) and processed with CellA Imaging Software (Olympus Soft Imaging Solutions GmbH, Germany) and open-source applications CellX and Image J.

Shelukhina I., Spirova E., Kudryavtsev D., Ojomoko L., Werner M., Methfessel C., Hollmann M, & Tsetlin V. (2017). Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants. PLoS ONE, 12(8), e0181936.

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HER2 Gene Copy Number Quantification

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FISH was performed by using a PathVysion HER2 DNA Probe Kit (Vysis, Downers Grove, IL, USA) according to the manufacturer’s instructions. Then, the HER2 gene copy number was quantified by using an epifluorescence microscope (Olympus, Tokyo, Japan). At least 60 tumor cell nuclei from three separate regions were used to measure signals from DNA probes specific for HER2 and the centromeric region of chromosome 17 (CEP17). HER2 gene amplification was determined according to ASCO/CAP guidelines [21 (link)]. Specifically, HER2 negativity was defined as a dual-probe HER2/CEP17 signal ratio less than 1.8 with an absolute HER2 gene copy number of less than 4 signals per cell. On the other hand, a HER2/CEP17 ratio greater than 2.2 with an absolute HER2 gene copy number of greater than 6 signals per cell was considered to be HER2 positive. HER2/CEP17 ratios between 1.8 and 2.2 or absolute HER2 copy numbers between 4 and 6 were considered to be HER2 equivocal.

Kim S., Lee Y, & Koo J.S. (2015). Differential Expression of Lipid Metabolism-Related Proteins in Different Breast Cancer Subtypes. PLoS ONE, 10(3), e0119473.

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Measuring NF-κB Translocation by Immunofluorescence

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Immunofluorescence assay were used to measure NF-κB p65 subunit nuclear translocation according to previously published methods (He et al., 2014, Pacurari et al., 2012 (link)). Briefly, the cells were grown on cover slips and treated with LPS, MWCNT, or combination for 24 h. After the treatments, the cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% TritonX-100/PBS. Cells were incubated with 5% bovine serum albumin (BSA)/PBS/0.1% TritonX-100 for 2 h at room temperature and then incubated with anti-NF-κB p65 antibody (Abcam, Cambridge, MA) in 5%BSA/PBS/0.1%TritonX-100 at 1:1000 dilution overnight at 4°C. After primary incubation, the cells were washed three times with PBS and incubated with an Alexa-Fluor^®-488-conjugated secondary antibody (Molecular Probes, Life Technologies, MA)/5% BSA/PBS/0.1%TrtionX-100 at a dilution of 1:1000 for an hour in the dark. After secondary antibody incubation, the cells were washed with PBS three times for 5 min each time, and then mounted with a mounting antifade solution containing DAPI (Molecular Probes,). Images were taken with an Olympus epifluorescence microscope.

Pacurari M., May I, & Tchounwou P. (2016). Effects of lipopolysaccharide, multiwalled carbon nanotubes, and the combination on lung alveolar epithelial cells. Environmental toxicology, 32(2), 445-455.

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Quantifying Discontinuous Tight Junctions in iPSC-BMECs

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Immunocytochemistry was conducted on iPSC-BMECs following mono/co-culture conditions and on all experimental groups utilized in co-culture experiments as previously described (Stebbins et al. 2015 ). Primary antibody sources and dilutions are provided in Supplementary Table 1. Cells were fixed in cold methanol (100%, Sigma) for 15 min. Cells were blocked in 10% goat serum (Sigma) for 30 min at room temperature. Images were taken on Olympus epifluorescence microscope. Primary antibodies, dilution ratios, fixation and blocking agents were previously described (Stebbins et al. 2015 ). Discontinuous tight junctions were quantified in occludin and claudin-5 immuno-labeled BMECs following monoculture or co-culture with iPSC-derived neurons and astrocytes (1:3 ratio). Following immunostaining with occludin or claudin-5, cells that lacked at least one continuous junction were classified as discontinuous. Images were processed in Image J with a minimum of ten fields with approximately 30 cells/field from three separate differentiations were quantified and all experimental groups remained blinded until completion of the study. Using the same images, the area of each image that exhibited occludin or claudin-5 immunoreactivity was measured to determine the area fraction index.

Canfield S.G., Stebbins M.J., Morales B.S., Asai S.W., Vatine G.D., Svendsen C.N., Palecek S.P, & Shusta E.V. (2017). An Isogenic Blood-Brain Barrier Model Comprising Brain Endothelial Cells, Astrocytes and Neurons Derived from Human Induced Pluripotent Stem Cells. Journal of neurochemistry, 140(6), 874-888.

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