The attachment of the cells to scaffolds and cell morphology were observed using immunofluorescence staining (IF) or SEM (described above). SAP-based hydrogels were prepared as described above. DPSCs were seeded on the surface of hydrogels (2 × 104 cells per well, n=3). After 3 days, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Fluorescent phalloidin and DAPI (Sigma) were used to label F-actin and nuclei, respectively. The images were captured using a Leica TCS SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany).
Fluorescent phalloidin
Manufactured by Merck Group
Sourced in United Kingdom
Fluorescent phalloidin is a fluorescent dye that selectively binds to actin filaments in cells. It is commonly used in microscopy applications to visualize the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 2 confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Ab5535, by Merck Group (1 mentions)
Bu 33, by Merck Group (1 mentions)
Lsm51o laser confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Lsm 510 confocal microscope, by Leica (1 mentions)
α tubulin clone dm1a, by Merck Group (1 mentions)
Dmlb microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
Antiphospho histone h3 s10, by Abcam (1 mentions)
Eclipse e1000, by Nikon (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
8 protocols using fluorescent phalloidin
1
DPSC Adhesion and Morphology on SAP Hydrogels
2
Apoptosis Analysis in Chicken Heart
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Hearts were dissected free from chicken embryos and fixed using cold 4% PFA o/n; 100‐μm‐thick vibratome sections were obtained and specimens analyzed for apoptotic DNA fragmentation by the terminal deoxynucleotidyl transferase‐mediated dUTP‐TRIC nick end labeling (TUNEL) assay. We also employed TUNEL analysis on cell dissociates after immunolabeling. We followed the manufacturer's instructions for the in situ cell death detection kit (Roche). Counterstaining using fluorescent phalloidin (Sigma) was also performed. Samples were analyzed by confocal microscopy as described. Each image in this work is representative of at least three independent experiments.
3
Cellular Analyses of Limb Development
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The β-galactosidase activity assay20 (link) was performed at pH 6 in vibratome sections of limb autopods fixed in 4% glutaraldehyde.
Neutral red staining, TUNEL assay, and electron microscopy were performed as described previously2 (link).
Immunolabeling was performed in limb tissue samples fixed in 4% PFA. We employed both squashed interdigital tissue fragments or vibratome sections permeabilized with Triton X-100 in PBS. The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich). Counterstaining to distinguish nucleus and cytoplasm was performed using fluorescent-phalloidin (Sigma Aldrich) or DAPI (Vector Laboratories). Observation were made with a LSM51O laser confocal microscope (Zeiss).
Neutral red staining, TUNEL assay, and electron microscopy were performed as described previously2 (link).
Immunolabeling was performed in limb tissue samples fixed in 4% PFA. We employed both squashed interdigital tissue fragments or vibratome sections permeabilized with Triton X-100 in PBS. The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich). Counterstaining to distinguish nucleus and cytoplasm was performed using fluorescent-phalloidin (Sigma Aldrich) or DAPI (Vector Laboratories). Observation were made with a LSM51O laser confocal microscope (Zeiss).
4
Immunostaining of Cytoskeletal Structures
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Cells grown on glass coverslips were immunostained with primary α-tubulin (clone DM1-A, Sigma), SEPT2 (Atlas), GFP (Cell Signaling) or V5 (Invitrogen) antibodies, after fixation with 3.75% paraformaldehyde/PBS, permeabilization with 0.1% Triton X-100/PBS and blocking with 0.5% BSA/PBS. Secondary antibodies were Alexa Fluor 488- or 555-conjugated antibodies against rabbit or mouse IgGs (Molecular Probes, Invitrogen) and Cyanine 5 antibody against mouse IgGs (Abcam, Paris, France). Actin microfilaments were visualized by fluorescent phalloidin (Sigma).
Images were acquired using either a Zeiss LSM 510 confocal microscope (63 × 1.4 NA objective) or a Leica DMLB microscope (40 or 100 × 1.3 NA objective). In the latter case, image acquisitions were performed using a Scion CFW1312M CCD camera driven from an Apple Macintosh G4 computer and homemade software. Data were quantified using the ImageJ software.
Images were acquired using either a Zeiss LSM 510 confocal microscope (63 × 1.4 NA objective) or a Leica DMLB microscope (40 or 100 × 1.3 NA objective). In the latter case, image acquisitions were performed using a Scion CFW1312M CCD camera driven from an Apple Macintosh G4 computer and homemade software. Data were quantified using the ImageJ software.
5
Fluorescent Imaging of Cytoskeleton in HUVECs
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The treated HUVECs were fixed with 4% paraformaldehyde and incubated with fluorescent phalloidin (Sigma) according to the manufacturer’s procedure. Following staining with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Switzerland), the HUVECs were mounted with an anti-fluorescence quenching agent and observed under a CLSM (SP5, Leica).
6
Wound Healing Assay for Cell Migration
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To analyse cell migration, the wound-healing technique was used. Briefly, confluent EC monolayers on a tissue culture dish were wounded by manually scratching with a pipette tip after an overnight starving, washed with starving medium and incubated at 37 °C for 8 h in complete media containing Mitomycin C (4 μg ml−1). The wound-induced cell migration was followed by staining with fluorescent phalloidin (10 μM, Sigma).
7
Immunofluorescence Staining of Cells
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Cells, grown on glass coverslips slides in 24 mm Petri dishes, were fixed in 4% paraformaldehyde at room temperature for 10 minutes, permeabilized with 0.2% Triton X-100 at room temperature for 5 minutes, washed with PBS and then stained with fluorescent phalloidin (Sigma Aldrich) or Vimentin (Abcam) followed by the secondary antibody anti-mouse Alexa 488 (Molecular Probes, Eugene, OR). Slides were fitted with Fluoroshield mounting medium with DAPI (Abcam) and analyzed with a fluorescence microscope [images were recorded with a Spot Insight digital camera (Delta Sistemi, Rome, Italy) equipped with a system of image analysis (IAS 2000; Delta Sistemi)].
8
Cytoskeleton and Chromatin Analysis
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The sulforhodamine B (SRB) proliferation assay (Sigma-Aldrich) and immunofluorescence experiments were performed as previously described (Caccia et al. 2010) (link). PTC and ATC cells were stained with fluorescent phalloidin (Sigma-Aldrich) for visualizing the cytoskeleton, with diamidino-2-phenylindole (DAPI) (Biostatus Limited, Leicestershire, UK) as a nuclear marker, with anti-b-tubulin (Sigma-Aldrich) for visualizing microtubules organization and with anti-phospho-histone H3 (S10) (Abcam, Inc.) for visualizing histone H3. Slides were imaged with immunofluorescence microscopy (Eclipse E1000; Nikon Instruments, Inc., NY, USA).
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