Following the MTS assay, the cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min. The morphology of the cells was analyzed via the staining of the actin filaments of the cells with phalloidin. For endothelial cell maturation, the von Willebrand factor was immunostained. In both cases, the samples were permeabilized with 1% of BSA in PBS containing 0.1% Triton X-100 for 20 min, and treated with 1% of Tween for 20 min at RT. The samples were then incubated with Atto 488-conjugated phalloidin (1:500; Sigma-Aldrich) for 20 min at RT. Alternatively, the samples were incubated with rabbit anti-von Willebrand primary antibody (1:400, Sigma-Aldrich, Merk, Darmstadt, Germany, Cat. No. F3520) overnight at 4 ◦C. After the samples had been rinsed twice with PBS, they were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:400). Images of the stained cells were captured under an IX-50 microscope (objective x 10) equipped with a DP 70 digital camera (both from Olympus, Tokyo, Japan). The intensity of immunofluorescence staining for von Willebrand factor in HSVECs was evaluated using the ImageJ software (ImageJ.org) and normalized per well.
Atto 488 conjugated phalloidin
Manufactured by Merck Group
Atto 488-conjugated phalloidin is a fluorescent probe used for labeling and visualizing F-actin in cells. It binds to the actin filaments, allowing the detection and analysis of the cellular cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Ix50 microscope, by Olympus (2 mentions)
Dp70 digital camera, by Olympus (2 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Nonidet np 40, by Merck Group (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Pbs tablet, by Merck Group (1 mentions)
γ globulin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Trypsin edta, by Lonza (1 mentions)
Sp8 confocal multiphoton microscope, by Leica (1 mentions)
Alexa 647 conjugated mouse anti β catenin, by Cell Signaling Technology (1 mentions)
Vectorshield containing dapi, by Vector Laboratories (1 mentions)
Metavue imaging software, by Molecular Devices (1 mentions)
Alexa fluor 594 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using atto 488 conjugated phalloidin
1
Endothelial Cell Maturation Assay
2
Synthesis and Characterization of PEGylated UPy Compounds
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PCLdiUPy
and PEG10KdiUPy were
synthesized by SyMO-Chem BV (Eindhoven, The Netherlands). The syntheses
of PEG2KdiUPy25 (link) and MeOPEG5KUPy27 (link) were described previously.
PBS tablets, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), EDTA,
non-idet NP-40, gelatin from porcine skin, bovine serum albumin (BSA),
fibrinogen from bovine plasma, γ-globulins from bovine blood,
mowiol 4-88, anti-human vinculin mouse IgG1 antibody (V9131), ATTO
488 conjugated phalloidin, and 4′,6-diamidino-2-phenylindole
(DAPI) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands).
EGM-2 Bulletkit, penicillin/streptomycin, and trypsin/EDTA were purchased
from Lonza (Breda, Netherlands). Alexa 555 conjugated goat anti-mouse
IgG1 was purchased from Molecular Probes. Tris, NaCl, and Triton X-100
were purchased from Merck Millipore, fetal bovine serum (FBS) from
Greiner Bio, and Dulbecco’s Modified Eagle Medium (#41966)
from Gibco Life Sciences. BSA, fibrinogen, and γ-globulins were
obtained as powders and used without further purification.
and PEG10KdiUPy were
synthesized by SyMO-Chem BV (Eindhoven, The Netherlands). The syntheses
of PEG2KdiUPy25 (link) and MeOPEG5KUPy27 (link) were described previously.
PBS tablets, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), EDTA,
non-idet NP-40, gelatin from porcine skin, bovine serum albumin (BSA),
fibrinogen from bovine plasma, γ-globulins from bovine blood,
mowiol 4-88, anti-human vinculin mouse IgG1 antibody (V9131), ATTO
488 conjugated phalloidin, and 4′,6-diamidino-2-phenylindole
(DAPI) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands).
EGM-2 Bulletkit, penicillin/streptomycin, and trypsin/EDTA were purchased
from Lonza (Breda, Netherlands). Alexa 555 conjugated goat anti-mouse
IgG1 was purchased from Molecular Probes. Tris, NaCl, and Triton X-100
were purchased from Merck Millipore, fetal bovine serum (FBS) from
Greiner Bio, and Dulbecco’s Modified Eagle Medium (#41966)
from Gibco Life Sciences. BSA, fibrinogen, and γ-globulins were
obtained as powders and used without further purification.
3
Immunostaining of BME-embedded Organoids
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BME droplets containing organoids were carefully collected into 1.5 ml tubes and spun down in a tabletop centrifuge at 600 × g for 5 min. The supernatant and visible fraction of attached BME were removed. The pellet was resuspended in 4% paraformaldehyde and fixed at room temperature for 15–20 min. Fixed organoids were washed 3 times in 1x PBS at 10–15-min intervals. The organoids were blocked and permeabilized in a solution containing 5% DMSO, 0.5% Triton-X-100 (Sigma, T8787), and 2% normal donkey serum (Sigma, D9663) for one hour at 4 °C. The samples were stained overnight with Alexa 647-conjugated mouse anti-β-catenin (1:200; Cell Signaling Technology, 4627S) and ATTO 488-conjugated phalloidin (1:300; Sigma, 49409-10NMOL). The samples were washed three times with 1x PBS. During the last wash, the samples were incubated with 2 μg/ml DAPI before mounting on coverslips in a solution containing 60% glycerol and 2.5 M fructose11 (link), followed by imaging on a multiphoton SP8 confocal microscope (Leica).
4
Immunofluorescence Staining of V5-tagged Cells
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Cells fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS for 20 minutes at RT, then washed with PBS containing 0.1% (v/v) Tween 20 three times and blocked with 1% (w/v) bovine serum albumin, 0.1% (v/v) Triton X-100 in PBS containing 0.1% Tween 20 for 1 hour. Cells were incubated with primary antibodies, mouse monoclonal anti-V5 epitope or the appropriate control IgGs, diluted in the blocking buffer overnight at 4°C. Cells were washed with PBS containing 0.1% (v/v) Tween 20 for 10 minutes three times and incubated with Alexa Fluor 594-conjugated antibodies (Invitrogen) and Atto 488-conjugated phalloidin (Sigma) diluted with the blocking buffer for 1 hour at room temperature, protected from light. Stained cells were washed with PBS containing 0.1% (v/v) Tween 20 for 10 minutes three times and mounted using Vector Shield containing DAPI (Vector Laboratories). Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software (Molecular Devices).
5
Phalloidin Staining and Quantification
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Phalloidin staining was performed according to Craig and Avasthi (2019) (link). Cells were adhered to coverslips for 2 min and were fixed with fresh 4% PFA in 7.5 mM Hepes in 1× PBS for 15 min, washed in 1× PBS for 3 min, permeabilized in cold (−20°C) 80% acetone for 5 min and then 100% cold acetone, and allowed to air-dry. Coverslips were rehydrated in 1× PBS for 5 min and incubated with Atto 488–conjugated phalloidin (49409-10Nmol; Sigma-Aldrich) for 16 min, and then washed in 1× PBS for 5 min, covered (Craig & Avasthi, 2019 (link)). Coverslips were air-dried, mounted with Fluoromount-G, and imaged at 100× on the spinning disk confocal microscope. For dot quantification, maximum-intensity projections were generated for each image stack and dots per cell counted using FIJI’s cell counter plugin.
6
Phalloidin Staining of Adherent Cells
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Following the MTS assay, the cells were rinsed with phosphatebuffered saline (PBS) and were fixed with 4% paraformaldehyde in PBS for 20 min. The initial cell adhesion and cell morphology were analyzed by staining the cells with phalloidin after 1 day in culture. In addition, cells that had been cultured for 7 days were stained with phalloidin. The samples were incubated with Atto 488-conjugated phalloidin (1 : 500; Sigma-Aldrich) and were counterstained with Hoechst 33258 (5 µg ml -1 in PBS, Sigma-Aldrich) for 20 min at room temperature (RT). Images of the adherent cells were captured under an IX-50 microscope equipped with a DP 70 digital camera (both from Olympus) and under a Dragonfly 503 spinning disk confocal microscope equipped with a Zyla 4.2 PLUS sCMOS camera (Andor, Belfast, UK).
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