To achieve large hiPSC colonies, 2 × 103 cells were seeded per one well of a vitronectin-coated 6-well plate. Cells were expanded for 5–7 days with daily media change to induce large hiPSC colonies. After expansion, hiPSCs were washed with PBS and fixed with 4% paraformaldehyde. Cells were permeabilized using 0.1% triton X-100 (Biosesang) for 10 minutes. After permeabilization, cells were blocked for 30 minutes in RT with PBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) (PBA). Primary antibodies were diluted in PBA with the following dilution ratios; OCT4 (1/100; Santa Cruz Biotechnology, Dallas, TX, USA), KLF4 (1/250; Abcam, Cambridge, UK), SOX2 (1/100; BioLegend, San Diego, CA, USA), TRA-1-60 (1/100; EMD Millipore), TRA-1-81 (1/100; EMD Millipore), and SSEA4 (1/200; EMD Millipore). Primary antibodies were incubated for 2 hours at RT. Alexa Fluor 594- (1/400; Life Technologies) and 488-conjugated secondary antibodies (1/400; Life Technologies) were diluted in PBA and incubated for 1 hour at RT avoiding light. Cells were washed and mounted using ProLong Antifade mounting reagent (Thermo Fisher Scientific, Waltham, MA, USA). Stained colonies were detected with an immunofluorescence microscope.
Prolong antifade mounting reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Antifade mounting reagent is a glycerol-based aqueous solution designed for use in fluorescence microscopy. It is formulated to reduce photobleaching of fluorescent dyes, allowing for longer observation times and improved image quality.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Ssea 4, by Merck Group (3 mentions)
Tra 1 60, by Merck Group (3 mentions)
Tra 1 81, by Merck Group (3 mentions)
Triton x 100, by Biosesang (3 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (3 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Phalloidin ifluor 594, by Abcam (2 mentions)
Zen 2011, by Zeiss (2 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Poly l lysine coated slide, by Thermo Fisher Scientific (1 mentions)
Orca ccd camera, by Hamamatsu Photonics (1 mentions)
Nmnat3, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
Dm5500b microscope, by Leica (1 mentions)
Dm5500b, by Leica (1 mentions)
Operetta high content screening system, by PerkinElmer (1 mentions)
Ethanol, by Merck Group (1 mentions)
18 protocols using prolong antifade mounting reagent
1
Immunofluorescence Characterization of hiPSC Colonies
2
Immunofluorescence Analysis of Cytoskeletal Dynamics
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Control and SEPT7-depleted epithelial cells cultured on collagen-coated coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton-X100 at room temperature. Fixed samples were blocked for 60 min in HEPES-buffered Hanks’ balanced salt solution (HBSS) containing 1% bovine serum albumin, followed by a 60-min incubation with anti-α-tubulin antibody. Samples were then washed and incubated with Alexa-Fluor-488–conjugated phalloidin and Alexa-Fluor-555–conjugated donkey anti-mouse secondary antibodies for 60 min, rinsed with blocking buffer, and mounted on slides with ProLong Antifade mounting reagent (Thermo-Fisher). Immunolabeled cell monolayers were imaged using a Leica SP8 confocal microscope (Wentzler, Germany). The Alexa Fluor 488 and 555 signals were acquired sequentially in frame-interlace mode, to eliminate cross talk between channels. Images were processed using Adobe Photoshop. The images shown are representative of at least three experiments, with multiple images taken per slide.
3
Immunostaining of Expanded iPSC Colonies
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iPSCs (1 × 103 cells/well) were plated in a vitronectin-coated 12-well plate with Y-27632 dihydrochloride (10 uM/ml). To induce the formation of large iPSC colonies, the cells were expanded for 5–7 days with daily media changes. After expansion, iPSCs were washed with PBS, fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100 (Biosesang, Seoul, Korea) for 10 min, and then blocked for 30 min at RT with PBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) (PBA). The cells were incubated with primary antibodies for 2 h at RT (manufacturers and dilution factors of primary antibodies are provided in Supplementary Table S3 ) and then with Alexa Fluor 594- (1/400; Life Technologies) or 488-conjugated secondary antibody (1/400; Life Technologies) for 1 h at RT. The cells were washed and mounted using ProLong Antifade mounting reagent (Thermo Fisher Scientific)31 (link). Stained colonies were detected with a fluorescence microscope.
4
Immunofluorescence Visualization of Cofilin
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Jurkat cells were adhered on poly-l -lysine coated slides (Thermo scientific), fixed with 3.7% paraformaldehyde. Cells were then permeabilized with 0.1% Triton X-100, washed and incubated overnigh with PBS supplemented with 1% bovine serum albumin (BSA) in the presence of anti-Cofilin antibodies. Following incubation with Alexa Fluor 594-conjugated goat anti-rabbit cross-adsorbed secondary antibodies (Thermo Fischer Scientific) for 30 min and nuclei staining with DAPI, coverslips were mounted in ProLong antifade mounting reagent (Thermo Fisher Scientific). Slides were then analyzed on a Axiovert 200M microscope (Zeiss) equipped with Orca CCD camera (Hamamatsu).
5
Immunofluorescence Characterization of Stem Cells
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The cells were washed twice with PBS and fixed with 1 mL of 4% paraformaldehyde for 30 min. After washing, the cells were incubated for 10 min at room temperature using 1 mL of NH4Cl solution. The cells were permeabilized using 0.1% Triton X-100 for 10 min and blocked for 30 min at room temperature in PBS containing 2% bovine serum albumin (Sigma-Aldrich) (PBA). Consequently, the primary antibodies OCT4 (Santa Cruz Biotechnology, 1:100 dilution), SSEA-4 (EMD Millipore, 1:200), TRA-1-60 (EMD Millipore, 1:200), SOX2 (BioLegend, 1:100), TRA-1-81 (EMD Millipore, 1:100), KLF4 (Abcam, 1:250), and NMNAT3 (Santa Cruz Biotechnology, 1:100) were diluted with PBA and the cells were incubated for 2 h at room temperature. After washing with PBA, the cells were incubated with Alexa Fluor 594-conjugated or 488-conjugated secondary antibodies (Life Technologies) in the dark for 2 h. For staining the nuclei, 4′,6-diamidino-2-phenylindole was incubated for 20 min at room temperature. The cells were mounted using ProLong Antifade mounting reagent (Thermo Fisher Scientific) and analyzed by Leica immunofluorescence microscopy.
6
Immunostaining of hiPSC Colonies
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To achieve hiPSC colonies, 2 × 103 iPSCs were seeded in one well of a vitronectin-coated 6-well plate for each staining. Cells were expanded for 5–7 days with E8 media which was changed daily. The staining process started with a wash with PBS and cells were fixed with 4% paraformaldehyde (Biosesang, Seongnam, Republic of Korea). After another wash with PBS, cells were permeabilized using 0.1% triton X-100 (Biosesang) for 10 min, then blocked for 30 min at room temperature (RT) with PBS supplemented with 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) (PBA). Primary antibodies were diluted in PBA and incubated for 2 h at RT. The following dilution factors were used in this experiment: SSEA4 (1:200; EMD Millipore, Billerica, MA, USA), TRA-1-60 (1:100; EMD Millipore), TRA-1-81 (1:100; EMD Millipore), OCT4 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), SOX2 (1:100; BioLegend, San Diego, CA, USA), KLF4 (1:250; Abcam, Cambridge, UK). After washing the cells, Alexa Fluor 594-(1:400; Life Technologies, Carlsbad, CA, USA) and 488-(1:400; Life Technologies) conjugated secondary antibodies were diluted in PBA and incubated for 1 h eat RT avoiding light. Cells were then washed and mounted using ProLong Antifade mounting reagent (Thermo Fisher Scientific). Colonies were detected under an immunofluorescence microscope.
7
Immunofluorescence Imaging of Cells
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Cell monolayers were grown on glass coverslips or collagen‐coated coverslips (10 μg/ml). After the indicated treatments, cells were washed in PBS, fixed in 4% PFA, permeabilized in PBS 0.3% Triton, and blocked in PBS 5% goat serum. Coverslips were then incubated overnight at 4°C with primary antibody diluted in PBS 5% goat serum. Following 1‐h incubation with Alexa Fluor‐conjugated secondary antibody, coverslips were mounted with Prolong antifade mounting reagent (Thermo Fisher Scientific). Nuclei were stained with Hoechst 33342 (Life Technologies). F‐actin was stained with Alexa Fluor 488 phalloidin (Fisher Scientific) or phalloidin‐iFluor 594 (Abcam) reagents. Coverslips were imaged using a wide‐field Leica DM5500B microscope.
8
Immunofluorescence Staining of MDA-MB-231 Cells
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MDA-MB-231 cell monolayers plated on collagen-coated coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 at room temperature. Fixed cells were blocked for 60 min in PBS containing 1% bovine serum albumin. Cells were incubated with the appropriate concentrations of primary antibodies in blocking solution for 60 min, washed with blocking buffer, incubated with Alexa dye-conjugated secondary antibodies and Alexa-labeled phalloidin (to detect filamentous (F) actin) for 60 min, washed, and mounted on slides with a ProLong Antifade mounting reagent (Thermo Fisher). Labeled cell monolayers were observed using a Zeiss LSM 700 Laser Scanning Confocal Microscope (Carl Zeiss Microimaging Inc.; Thornwood; NY). The Alexa Fluor 488 and 555 signals were imaged sequentially in frame-interlace mode to eliminate crosstalk between channels. Image analysis was conducted using imaging software ZEN 2011 (Carl Zeiss Microscopy Inc.) and Adobe Photoshop. Images shown are representative of at least three experiments. Multiple images were captured from each slide.
9
Quantifying Bacterial Adhesion and Viability
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A suspension of S. aureus in 0.9% saline at a concentration of 10 7 CFU/mL was prepared as described above. 1 mL of this suspension was placed on each 2.25 cm 2 SIPN coating (prepared on an a pristine glass slide and sterilized with ethanol). The control surface was a pristine glass slide, washed with ethanol and dried. The surfaces were placed in a petri dish, the dish was sealed with parafilm, and the samples were incubated for 24 hours at 37 °C at 75% humidity. The surfaces were then gently washed with 10 mL of distilled water, then incubated with a mixture of the LIVE/DEAD TM BacLight bacterial viability assay dyes (SYTO 9 and propidium iodide, Life Technologies, Burlington, Canada) for 30 minutes in the dark according to the manufacturer's directions. The surfaces were then gently washed with deionized water and treated with Prolong Antifade mounting reagent (ThermoFisher Scientific, Massachusetts, USA) and covered with a slip cover. The prepared surfaces were then imaged by laser fluorescence microscopy using an Upright Zeiss Axioimager Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) (Laser 488 nm for the SYTO 9 with a pass filter of 505-530 nm and a laser at 543 nm for the propidium iodide with a pass filter of 615 nm, magnification 40´). All the images were obtained and refined with the ZEN software. Each sample was tested in triplicate.
10
Immunofluorescence Staining of Cell Monolayers
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Cell monolayers were grown on glass coverslips or collagen-coated coverslips (10 μg/mL). After the indicated treatments, cells were washed in PBS, fixed in 4% PFA, permeabilized in PBS 0.3% Triton and blocked in PBS 5% goat serum. Coverslips were then incubated overnight at 4°C with primary antibody diluted in PBS 5% goat serum. Following 1 h incubation with Alexa-fluor conjugated secondary antibody, coverslips were mounted with Prolong antifade mounting reagent (ThermoFisher Scientific). Nuclei were stained with Hoechst 33342 (Life Technologies). F-actin was stained with Alexa Fluor 488 phalloidin (Fisher Scientific) or phalloidin-iFluor 594 (Abcam) reagents. Coverslips were imaged using a wide-field Leica DM5500B microscope.
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