Tenocytes cultured on coverslips or type I collagen pre-coated membranes were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 in PBS for 15 min at room temperature. After applying blocking buffer containing 5% FBS and 0.3% Triton X-100, cells were incubated first with anti-α-smooth muscle actin antibody (1:100, Abcam, Cambridge, UK, ab5694), and then with Goat Anti-Rabbit Alexa Fluor® 488 secondary antibody (1:200, Abcam, Cambridge, UK, ab150077). Lastly, to stain F-actin, cells were incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1:250, ThermoFisher, Waltham, MA, USA, A12380). Stained cells were mounted using DAPI-containing mounting reagent (Abcam, Cambridge, UK, ab104139), and images were collected using a fluorescence microscope (Zeiss AXIOVERT 200 M).
Tritc conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
TRITC-conjugated phalloidin is a fluorescent probe used to label and visualize filamentous actin (F-actin) in fixed cells. It binds selectively to F-actin and emits a red fluorescent signal when excited by appropriate wavelength light, allowing for the detection and localization of actin structures within the cell.
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Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Abcam (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dmi4000b fluorescence microscope, by Leica (1 mentions)
Las af computer software, by Leica (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Ix81 inverted fluorescence microscope, by Olympus (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
21 protocols using tritc conjugated phalloidin
1
Immunofluorescence Staining of Tenocytes
2
Visualizing Immune Cell Interactions
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NGFR‐enriched CD4OT‐II (0.25 × 106 cells) were either not labeled or labeled with CFSE (Thermo Fisher Scientific). The cells were then mixed with CMAC‐labeled B cells (0.5 × 106 cells) and incubated on PLL (0.1%, Sigma‐Aldrich)‐coated cover glasses in each well of a 24‐well plate for 1–2 h. Before incubation, the plate was spun at 1,000 rpm for 60 s to reduce the cell settling time. Sample was fixed using 1–3% PFA in PBS and incubated for another 10 min at RT. Cells were then permeabilized with 0.1% Triton X‐100 for 2–3 min and blocked in 1% BSA in PBS (v/v) for 30 min at RT. Following washing with 1× PBS, TRITC‐conjugated Phalloidin (Thermo Fisher), mouse anti‐p‐Tyr 100 (Cell Signaling Technology), and rat anti‐mouse IL‐4 (R&D Systems) was used for staining. Cover glasses were washed three times with 1× PBS and incubation with secondary anti‐mouse Alexa‐568 or anti‐Rat Alexa‐568 (1:2,000, in 1× PBS with 1% BSA, Invitrogen) was added and incubated for 1 h at RT. After washing with 1× PBS, cover glasses were mounted using Mowiol (Calbiochem), dried over night at RT protected from light and stored at 4°C until analyzed by epifluorescence (IX81 Olympus), confocal (TCS SP5), and spinning disc confocal (Nikon Ti PerkinElmer UltraVIEW) microscopy. Images were processed using ImageJ.
3
Visualizing Actin Cytoskeleton and Focal Adhesions
4
Visualizing Cortactin and H. pylori Vacuoles
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AGS wt and AGSΔcttn cells were grown and infected as described above. After 8 h of infection, the cells were fixed using 4% PFA for 10 min at 20 °C. After fixation, the cells were permeabilized with 0.25% Triton-X-100 for 10 min and stained with mouse α-cortactin antibodies (Merck-Millipore, #05-180) followed by adding the secondary, FITC-conjugated goat-anti-mouse antibody. In addition, cells were counterstained with TRITC-conjugated phalloidin (Thermo Fisher Scientific, Darmstadt, Germany, #R415). Samples were analysed using a Leica DMI4000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany). In order to analyze H. pylori-induced vacuoles, infected AGS cells (24 h) were stained with neutral red (see below) and imaged under light microscopy with polarizing contrast. Images were processed using the LAS AF computer software (Leica Microsystems).
5
Investigating HCEC Migration with rhGM-CSF
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An in vitro migration assay to investigate the migration rate of HCECs was performed using ibidi Culture-Inserts (ibidi GmbH, Martinsried, Germany) according to the manufacturer’s instructions. HCECs were seeded into the Culture-Inserts and grown to confluency. Growth factor-starved HCECs were treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). The Culture-Inserts within the cell plates were then removed to create a linear scratch wound. Images of the scratch wound fields were captured with an inverted light microscope (Eclipse TE300; Nikon, Tokyo, Japan) equipped with a digital camera at 0, 24, and 48 h of incubation. HCECs were stained with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Life technologies, Carlsbad, CA). The migration rate was defined as the ratio of the difference between the initial wound area and the remaining wound area vs. the initial wound area. The remaining wound areas photographed at 24 and 48 h were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Experiments were performed at least three times.
6
Morphological Analysis of MSCs on Fibers
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To examine the cell morphology on the fiber sheets, MSCs attached to fibers for 24 h were fixed in 10% formalin and then incubated with TRITC-conjugated phalloidin (1:1000; Life Technologies), before mounted with slow-fade gold fluorescent mounting medium containing DAPI (Life Technologies). Images of stained cells were captured using an Olympus IX81 inverted fluorescence microscope.
7
Osteoblast Morphology and Cytoskeleton Analysis
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The morphology and cytoskeletal arrangement of osteoblasts seeded onto the samples were examined by confocal laser scanning microscope (CLSM) and SEM after culturing for 12 hours.
The cells cultured on the samples were fixed with 2.5% glutaraldehyde in 0.1 M PBS buffer (pH =7.4) for 20 minutes, and rinsed three times with PBS. Then, each sample was dehydrated with graded alcohol (10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%) and finally with absolutely pure ethyl alcohol, and the samples were dried for SEM analysis to enable the morphology of osteoblasts to be studied.
The cells were washed twice with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich). Then, they were permeabilized with 0.1% Triton-X for 1 hour. The cells were incubated for 1 hour with TRITC-conjugated phalloidin (Molecular Probes, Eugene, OR, USA) at room temperature in the dark. The final step was to stain the cell nuclei with DAPI (Dojindo Laboratories) for 10 minutes at room temperature. The cytoskeletal arrangement of the cells was obtained with a CLSM (LSM-510, Carl Zeiss, Jena, Germany).
The cells cultured on the samples were fixed with 2.5% glutaraldehyde in 0.1 M PBS buffer (pH =7.4) for 20 minutes, and rinsed three times with PBS. Then, each sample was dehydrated with graded alcohol (10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%) and finally with absolutely pure ethyl alcohol, and the samples were dried for SEM analysis to enable the morphology of osteoblasts to be studied.
The cells were washed twice with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich). Then, they were permeabilized with 0.1% Triton-X for 1 hour. The cells were incubated for 1 hour with TRITC-conjugated phalloidin (Molecular Probes, Eugene, OR, USA) at room temperature in the dark. The final step was to stain the cell nuclei with DAPI (Dojindo Laboratories) for 10 minutes at room temperature. The cytoskeletal arrangement of the cells was obtained with a CLSM (LSM-510, Carl Zeiss, Jena, Germany).
8
Quantifying Cell Morphology Changes
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After cells (1.4 × 104 cells per well in a 96-well culture plate) were incubated with fresh culture media containing small molecules (HS-104, HS-173) at a final concentration of 5 μM for 48 hours, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature. After permeabilization step with 0.1% Triton X-100 in PBS for 5 min, cells were stained with TRITC-conjugated phalloidin (Molecular Probes, OR, USA). Cells were imaged using an IN Cell Analyzer 2000 (GE Healthcare, UK). The cell shape index (CSI) of each cellular contour was calculated based on the ratio of cell width to cell length using NIH ImageJ.
9
Cytoskeleton and Nuclei Staining
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Transfected cells were fixed with 4% paraformaldehyde. After washing with PBS, the fixed cells were incubated with 100 nM TRITC-conjugated phalloidin (Molecular Probes, Eugene, USA) and 100 nM Dapi solution in PBS for 30 minutes in the dark. Staining was performed using fluorescence microscopy.
10
Actin Cytoskeleton Disruption Assay
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