Immunofluorescence was performed on OECs. The cells were fixed with 4% formalin solution at room temperature for 5 min then washed with PBS and permeabilized with 0.1% Triton X-100. The cells were then blocked with 1% bovine serum albumin (10082-139; Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min and incubated for 18 h at 4°C with anti-p75NGFR (1:500; AB1554; EMD Millipore) and anti-BrdU (1:200; mAb #5292; Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies. Following washing with PBS, the cells were incubated at 37°C for 2 h with respective anti-Mouse IgG H&L (Alexa Fluor 647; 1:500; ab150115; Abcam, Cambridge, MA, USA). Following incubation, the slides were washed with PBS, mounted and examined under a fluorescence microscope (Leica TCS SP2; Leica Microsystems GmbH, Wetzlar, Germany). Fluoroshield mounting medium with DAPI (Sigma-Aldrich; Merck KGaA) was used for DAPI staining and the staining was performed according to the manufacturer's protocol.
Fluoroshield mounting medium with dapi
Manufactured by Merck Group
Sourced in United States, Germany
Fluoroshield mounting medium with DAPI is a laboratory reagent used to mount and preserve fluorescently-labeled samples for microscopy. It contains the DNA-binding dye DAPI, which allows for the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Volocity software 6, by PerkinElmer (2 mentions)
Tcs sp5 2 confocal microscope, by Leica (2 mentions)
Alexa fluor 488, by Abcam (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Poly lysine coated slide, by Merck Group (2 mentions)
Lsm800 point scanning confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab1554, by Merck Group (1 mentions)
Anti mouse igg h l alexa fluor 647, by Abcam (1 mentions)
Ab150115, by Abcam (1 mentions)
Tcs sp2, by Leica (1 mentions)
Ab18058, by Abcam (1 mentions)
Ab39361, by Abcam (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
N cad, by BD (1 mentions)
Alexa fluor 555, by Merck Group (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Fv1000 scanning confocal microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
34 protocols using fluoroshield mounting medium with dapi
1
Immunofluorescence Analysis of OEC Cells
2
Immunostaining of Fibroblast Cytoskeletal Components
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WT and HOM fibroblasts were cultured for 48 h (±2 h) in untreated/treated conditions, fixed for 10 min with 4% formaldehyde in PBS at room temperature (RT), and processed as previously reported [45 (link)]. Briefly, the primary antibody mix was composed of GDB buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4), Alexa Fluor™ 647 Phalloidin (A22287, 1:40, Thermo Fisher) to stain the actin fibres (F-actin), and the following primary antibodies: anti-NBR1 (1:200; PA-30085, Thermo Fisher); anti-Vinculin (1:100; VINC; ab18058, Abcam, Cambridge, UK); anti-N-CADherin (1:200; N-CAD; 610921, BD Transduction Laboratories, Franklin Lakes, NJ, USA); anti-YAP1 (1:200; ab39361, Abcam). Then, samples were incubated for 1 h at RT with the appropriate secondary antibody, Alexa Fluor 488 (anti-mouse; Cat#A21202) or Alexa Fluor 555 (anti-rabbit; Cat#A31570), diluted 1:100 in GDB buffer and mounted using Fluoroshield mounting medium with DAPI (F6057, Sigma Aldrich).
3
Dox-Loaded Sphere Uptake in Cells
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SKBR3 and MSU1.1 cells (1 × 105 cells/well) were plated on 8-well Lab-Tek chambered coverslips (Nunc, Naperville, IL) and cultured for 24 h. Next, 10 μg/mL of Dox-loaded H2.1MS1:MS2, H2.1MS1:DOXMS2, and MS1:DOXMS2 spheres were added to the cells, which were incubated at 37 °C for 15 or 30 min. After washing with PBS, the cells were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA). Subsequently, the cells were washed with PBS and immersed in Fluoroshield mounting medium with DAPI (Sigma, St. Louis, MO) and then analyzed under an Olympus FV1000 scanning confocal microscope (Shinjuku, Tokyo, Japan) connected to a blue laser diode and an argon laser. Image acquisition and analysis were performed with a 60× objective, a 1.4 N.A. oil immersion lens, and FLUOVIEW Viewer software, ver. 4.1. The nuclei were visualized using 350 nm excitation and 440–480 nm emission wavelengths. To visualize the Dox-loaded spheres and the Dox released from the spheres, an excitation wavelength of 488 nm and an emission wavelength of 570–610 nm were used.
4
Confocal Imaging of Pseudomonas Infection in A549 Cells
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For confocal imaging, 2 ×105 A549 cells were seeded in 24-well plates containing 11 mm round glass coverslips and phosphate-free DMEM medium with 5% (v/v) FBS one day prior to infection. Infections with green fluorescence-labelled P. aeruginosa cells containing a σVreI-dependent red fluorescence transcriptional fusion were performed at a MOI of 10. At 0 and 10 hpi, the cells were fixated with 4% (v/v) paraformaldehyde in PBS. Samples were washed with PBS and coverslips were mounted on glass slides containing Fluoroshield mounting medium with DAPI (Sigma-Aldrich) to retain fluorescence and stain the A549 cells DNA. Confocal images were generated with a Nikon A1R confocal scanning laser microscope. NIS-Elements and ImageJ software were used to process the confocal images. Total corrected cellular fluorescence (TCCF) was calculated using the following equation: TCCF = integrated density − (area of selected cell × mean fluorescence of background readings), as described before56 (link),57 (link).
5
Cellular Uptake Dynamics of ATTO647N-labeled Silk Spheres
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To study cellular uptake, the SKBR3 cells were seeded onto 8-well Lab-Tek chambered cover glasses (Nunc, Naperville, IL, USA) at a density of 1×105 cells/well and then cultured for 24 h. Next, the cells were incubated with ATTO647N-labeled H2.1MS1:H2.1MS2 particles (10 µg/mL) for 4 h at 37 °C or 2 h at 4 °C. After washing with PBS, the cells were fixed with 4% paraformaldehyde (PFA; Sigma, St. Louis, MO, USA), and then the cell membranes were stained with ConA-FITC (Sigma, St. Louis, MO, USA) at a concentration of 50 μg/mL for 30 min. Subsequently, the cells were washed with PBS, immersed in Fluoroshield mounting medium with DAPI (Sigma, St. Louis, MO), and then analyzed under an Olympus FV1000 Confocal Laser Scanning Microscope (CLSM; Shinjuku, Tokyo, Japan) connected to a blue laser diode and an argon laser. Image acquisition and analysis were performed with a 60× objective, a 1.4 N.A. oil immersion lens and FLUOVIEW Viewer software, ver. 4.1. The DAPI-stained nuclei were visualized using 405 nm excitation and 440–480 nm emission wavelengths. Images of the silk spheres labeled with ATTO647N were visualized using 635 nm excitation and 575–675 nm emission wavelengths. To visualize cell membranes, 488 nm excitation and 495–525 nm emission wavelengths (FITC) were applied.
6
Immunofluorescence Imaging of Cells and Tissues
7
Retinal Oxidative Stress Visualization
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Retinal cryosections were incubated overnight at 4 °C with anti-mouse 4-hydroxynonenal (4-HNE) antibody (1:100; Cayman Chemical, Ann Arbor, Michigan). Slides were washed three times with 0.1% Triton X-100 in 0.1 M PBS (pH 7.4) followed by a 1 h incubation with goat anti-rabbit IgG-conjugated Alexa flour 488 secondary antibody (Molecular probes-Life Technologies, Grand Island, NY). Coverslips were mounted using fluoroshield mounting medium with DAPI (Sigma-Aldrich, St. Louis, MO) and images captured at 20X magnification using Zeiss Axioplan-2 imaging fluorescence microscope (Carl Zeiss, Göttingen, Germany).
8
Quantifying Tumor Hypoxia Using Pimonidazole
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Pimonidazole (Hypoxyprobe, Inc., Burlington, MA, USA) was injected intraperitoneally at the dose of 60 mg/kg body weight. The mice were sacrificed 1 h after the injection, and the tumor tissue was flash-frozen in Surgipath FSC 22 clear frozen section compound (Leica Biosystems Richmond, Inc., Richmond, IL, USA). The tissue was cryosectioned at a thickness of 7 μm and fixed in ice-cold acetone (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The sections were incubated with FITC-Mab1 (clone 4.3.11.3; Hypoxyprobe, Inc., Burlington, MA, USA) overnight at 4 °C to stain for Pimonidazole [39 (link)]. The slides were then mounted in Fluoroshield mounting medium with DAPI (Sigma-Aldrich, St. Louis, MO, USA) and stored at 4 °C. Images of three random fields of view (FOVs) were acquired for each sample using an upright fluorescence microscope (DM2500; Leica Microsystems, Wetzlar, Germany) and a color CCD (DP73; Olympus, MA, USA). ImageJ software was used to quantify the mean fluorescence intensity (MFI) [39 (link)], and statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test in GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).
9
Quantifying Immune Cells in Mouse Livers
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Fresh frozen livers of Atg7f/f and Lck-Cre Atg7f/f mice were sectioned on a cryostat in 8 μm thick sections. Sections were fixed in an ice-cold 75% acetone/25% ethanol mix and blocked in 2% normal goat serum and 3% bovine serum albumin (Sigma). Next, the sections were incubated overnight at 4°C with antibodies detecting CD68 and CD3e (both from Abcam). Subsequently, the sections were washed and incubated with antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 (both from Abcam). Nuclei were visualized using Fluoroshield mounting medium with DAPI (Sigma). T cells were manually quantified while blinded to the genotype using Fiji software. Three images were analyzed of every liver section and three liver sections were analyzed per mouse. Confocal images were acquired using a 20x objective on a Nikon TiE 2000 confocal microscope.
10
Immunohistochemical and Immunofluorescence Analysis of Stem Cell Markers in 3D Spheroids
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The expressions of PAX8, Axin2, ALDH1A1, and SOX9 were evaluated by IHC analysis. The FFPE spheroid sections (3 µm thick) were dewaxed in xylene and hydrated in graded alcohol. After antigen retrieval in sodium citrate buffer, the slides were incubated in overnight at 4°C with Abs at the following dilutions: 1:500 PAX8 (10336‐1‐AP; Proteintech), 1:200 Axin2 (20540‐1‐AP; Proteintech), 1:200 ALDH1A1 (15910‐1‐AP; Proteintech), and 1:500 SOX9 (HPA001758; Atlas Antibodies). Two gynecologic oncologists (S.S. and S.K.) independently evaluated the samples under a light microscope. Sections of normal endometria in the same patients were used as positive controls for all markers.
For immunofluorescence staining, FFPE spheroids or endometrium slides were loaded into a glass slide holder and dewaxed in xylene and then hydrated with alcohol. Sodium citrate buffer pH 6.0 was used for antigen retrieval in an autoclave for 10 min followed by incubation with primary Abs at a dilution of Axin2 1:200 and Ki‐67 1:100 overnight at 4°C followed by incubation with fluorescence‐labeled secondary Abs for 1 h at room temperature. Slides were then counterstained with Fluoroshield Mounting Medium with DAPI (Sigma‐Aldrich), and the immunofluorescence was detected using a Nikon Eclipse 50i fluorescence microscope with the appropriate filter.
For immunofluorescence staining, FFPE spheroids or endometrium slides were loaded into a glass slide holder and dewaxed in xylene and then hydrated with alcohol. Sodium citrate buffer pH 6.0 was used for antigen retrieval in an autoclave for 10 min followed by incubation with primary Abs at a dilution of Axin2 1:200 and Ki‐67 1:100 overnight at 4°C followed by incubation with fluorescence‐labeled secondary Abs for 1 h at room temperature. Slides were then counterstained with Fluoroshield Mounting Medium with DAPI (Sigma‐Aldrich), and the immunofluorescence was detected using a Nikon Eclipse 50i fluorescence microscope with the appropriate filter.
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