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37 protocols using glass chamber slide

1

Immunocytochemical Characterization of ECFCs

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For quality and purity control, each ECFC isolation was subjected to immunocytochemical characterization using antibodies against endothelial cell markers (CD31, von Willebrand factor (VWF)), fibroblast markers (CD90, TE-7) and muscle cell markers (smooth muscle actin (SMA), Desmin) as described previously.41 (link) In brief, cells were seeded on glass chamber slides (ThermoFisher Scientific) and fixed with acetone (Merck). TBE pH 8.0 (Gatt-Koller, Absam, Austria) with 0.1% Tween (Sigma-Aldrich) was used as rehydration and washing buffer. Primary antibodies (Supplementary Table 2) were applied for 30 min and visualization was performed with the Ultra Vision horseradish peroxidase Polymer Kit (ThermoFisher Scientific). Images were generated using a light microscope (Olympus BX53) with the UC90 camera (Olympus) and the corresponding cellSens Standard software (Olympus).
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2

Immunocytochemical Analysis of Cytochrome C

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Both SH-SY5Y cells and Glioma cells were plated on glass chamber slides (Thermo Fisher Scientific, Rochester, NY). Slides were pre-coated with poly-D-lysine for SH-SY5Y cells. After seeding, cells were allowed to attach for 1 day prior to treatment with Aβ in presence or absence of MTZ for 24 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde (10 min, RT), washed again, and blocked for 1 hour with 20 mg/ml BSA in PBS containing 0.3% Triton X-100 (PBST). Slides were further incubated with mouse monoclonal anti-CytC antibody (BD Biosciences; 1:200 in PBST containing 5 mg/ml BSA; 2h, RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, Grand Island, NY) 1:200 in PBST with 5 mg/ml BSA for 1h at RT, as previously described (Fossati et al., 2013 ). Fluorescence signals were visualized in a Zeiss LSM 510 laser scanning confocal/Confocor2 microscope using a 40x DIC oil immersion objective and LSM 510 software; acquired images were processed and analyzed using ImageJ (National Institute of Health; http://rsbweb.nih.gov/ij/).
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3

Visualizing Antimicrobial Biofilm Morphology

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To visualize the morphology of the antimicrobial treated biofilms grown on glass chamber slides (Thermo Scientific), the media was carefully removed without disturbing the biofilm. Contact mode AFM imaging in air was performed on a Nanosurf Easyscan 2 AFM (Nanosurf) using SHOCONG probes (APPNANO). Images were processed using Gwyddion software (Nečas and Klapetek).
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4

Quantifying Lipid Droplets in Cells

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Cells were grown on glass chamber slides (Thermo Fisher, Rochester, NY, USA) to 60% to 70% confluence, washed with PBS, and fixed with 4% (w/v) paraformaldehyde. Cells were washed with PBS and incubated at a 1:1000 dilution in PBS of a 1 mg/ml stock solution of Nile Red (Sigma-Aldrich) in acetone for 10 min at room temperature. Cell nuclei were counterstained and mounted with ProLong Gold Antifade reagent (Life Technologies, Grand Island, NY). A 40X LUMPLFL water immersion objective on an Olympus FV1000MPE multiphoton microscopy system (Olympus, Center Valley, PA) with a 690–1040 nm tunable MaiTai DeepSea femtosecond laser (Spectra-Physics, Santa Clara, CA) was used to acquire three dimensional (3D) image stacks from various fields of view in the sample. DAPI stained nuclei and Nile Red stained lipid droplets were simultaneously illuminated with incident laser light of 810 nm and detected at 455 nm (blue channel, DAPI) and 640 nm (red channel, Nile Red) respectively. The number and size of lipid droplets per cell were quantified using in- house software. Approximately 20–40 cells per field of view from at least six different fields of view were analyzed for each cell line.
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5

Isolation and Culture of Hematopoietic Stem Cells

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HSCs were isolated from WT and Cygb−/− mice using the pronase-collagenase digestion method as described previously66 (link) and were cultured on uncoated-plastic dishes (BD Falcon, Franklin Lake, NY, USA) or glass chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a 5% CO2/95% room air. Mouse hepatoma Hepa 1–6 cells (CRL-1830) obtained from American Type Culture Collection (Manassas, VA, USA) were maintained on uncoated-plastic culture plates (BD Falcon) in DMEM supplemented with 10% FBS and antibiotic.
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6

Immunostaining and Confocal Microscopy for PRIP-1 in HESCs

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Paraffin-embedded, formalin-fixed endometrial specimens were immunostained for PRIP-1 using the Novolink polymer detection system (Leica) as per manufacturer's instructions. Universal LSAB Plus kits (DAKO) were used as described previously (39 (link)) using primary antibodies against PRIP-1 (1:750 dilution; Sigma-Aldrich). As a negative control, the primary antibody was omitted and replaced by the corresponding IgG isotype. For confocal microscopy, primary HESCs were cultured on glass chamber slides (Thermo Scientific) and transfected with either NT or PRIP-1 siRNA. HESCs were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 in Tris-buffered saline with 0.05% Tween (TBS-T) for 30 minutes and blocked in 1% BSA in TBS-T for 1 hour. Endogenous proteins were stained with rabbit anti-FOXO1 (2880S, 1:100; Cell Signaling Technology), followed by antirabbit fluorescein isothiocyanate (F0205, 1:500; DAKO). Cells were mounted in Vectashield with 4′,6-diamidino-2-phenylindole (Vector Labs) and visualized under a Zeiss LSM 510 META confocal microscope.
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7

Immunostaining of Cells after Plasma Membrane Damage

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Cells were plated on 2-well or 4-well glass chamber slides (Thermo Fisher) coated with fibronectin (100 μg/mL in PBS, Millipore). Cells were maintained in complete media at 37°C and 5% CO2 in an environmentally controlled chamber. After PM damage, cells were fixed cells in 4% paraformaldehyde-PBS 10~20 min at room temperature, permeabilized with 0.3% Triton X-100 in 1X PBS, blocked in 3% BSA in 1X PBS, stained with primary antibodies for 2–4 hr (room temperature) to overnight at 4°C, followed by staining with 2° antibodies (1 hr) and DAPI stain. Samples were imaged using Lionheart microscope (Biotek).
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8

Immunocytochemistry of E-cadherin and α-catenin

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Cells were seeded into glass chamber slides (Thermo Scientific) pre-coated with fibronectin (5μg/mL; Santa Cruz Biotechnologies, Dallas, TX), grown until confluent, fixed with 2% paraformaldehyde, permeabilized (0.1% Triton X-100; Sigma) and blocked (PBS/1% BSA; 1hr) prior to incubation with anti-E-cadherin and/or anti-α-catenin primary antibodies (1:50; 1hr). Cells were labeled with goat-anti-mouse-IgG-AlexaFluor488 and/or goat-anti-rabbit-IgG-AlexaFluor594 (Life Technologies; 1:300; 1hr) before mounting with VectaShield+DAPI (Vector Laboratories, Burlingame, CA). Imaging was performed (60x) using an Olympus Provis AX70 microscope (Olympus, Richmond Hill, ON).
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9

Immunofluorescence Analysis of NRP-1 in BGC-823 Cells

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BGC-823 cells were plated onto glass chamber slides at a density of 1×104 cells/well (Thermo Fisher Scientific, Inc.) and cultured in RPMI-1640 medium at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. Following culture, cells were fixed with 4% paraformaldehyde at 37°C for 30 min and blocked with bovine serum albumin (BSA) at 37°C for 1 h. Cells were then incubated with anti-NRP-1 mAb (1:100; Cancer Research Centre, Medical College of Xiamen University) for 1 h at 37°C, followed by anti-mouse IgG tetramethylrhodamine (TRITC)-conjugated secondary antibodies (cat. no. ab6786; 1:1,000; Abcam) for 1 h at 37°C. After four washes in TBST, cells were stained with Hochest 33258 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 10 min and examined using a Zeiss LSM 710 confocal laser scanning microscope (Zeiss GmbH, Jena, Germany). Isotype control antibody of anti-NRP-1 mAb was used as the control (cat. no. ab81032; 1:1,000; Abcam).
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10

Virus-Mediated Cell Membrane Fusion Assay

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Virus-mediated cell membrane FFWI was performed as previously described (Guirakhoo et al., 1993 (link)). Essentially, C6/36 cells were seeded onto glass chamber slides (ThermoFisher, Waltham, MA) before infection with VEEV TC83 at an MOI of 1.0 and incubation at 28 °C/5%CO2 while maintaining the pH at 7.5. Twenty-four hours after infection, cells were incubated with various concentrations of MAb diluted in BA-1 for 2 h at 28 °C/5%CO2. After MAb treatment, cells were exposed to fusion medium (DMEM buffered at pH < 5.0 with 2-N-morpholinoethanesulfonic acid) for 1 h, after which fusion medium was replaced with DMEM at neutral pH. Cells were incubated another 24 h at 28 °C/5%CO2 before being fixed in absolute methanol and stained using the Wright-Giemsa stain kit (Abcam, Cambridge, MA). An isotype MAb (100 μg/ml), virus-infected non-treated cells at pH 7.5, and uninfected MAb-treated cells at pH 5.0 were included as controls. The number of nuclei and the number of cells in five microscopic fields (magnification 100-fold) were counted and the fusion index [1 - (number of cells/number of nuclei)] was calculated. A one-way ANOVA with Tukey’s multiple comparisons test was used to compare differences in percent fusion.
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