Akt2 knockdown and Akt2/MTSS1 double knockdown GEO cells plated on coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100/PBS for 15 min. Cells were stained with the F-actin probe Alexa Fluor 633 Phalloidin (Molecular Probes, Thermoscientific, San Diego, CA, USA) at 10 units/200 μl for 30 min and washed with PBS. After 4’-6 diamino-2-phenylindole (DAPI) staining to detect the nucleus, coverslips were mounted on slides using VECTASHIELD anti-fade mounting medium (Vector Laboratories, Burlingame, CA, USA). Cells were visualized using an LSM 710 laser scanning confocal microscope (Carl Ziess GmbH, Oberkochen, Germany).
Alexa fluor 633 phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Alexa Fluor 633 phalloidin is a fluorescent stain used for the detection and visualization of actin filaments in biological samples. It binds specifically to F-actin, allowing for the labeling and analysis of the cytoskeleton in cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Lsm 510 meta, by Zeiss (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Vectashield with dapi, by Vector Laboratories (3 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Methotrexate, by Merck Group (2 mentions)
Midi prepped endotoxin free plasmid dna, by Qiagen (2 mentions)
4 6 diamidino 2 phenylindole dapi dilactate solution, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Fv1200, by Olympus (2 mentions)
Lsm 780, by Zeiss (2 mentions)
N cadherin, by BD (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
35 mm glass bottom dishes, by MatTek (2 mentions)
78 protocols using alexa fluor 633 phalloidin
1
Visualization of Actin Cytoskeleton in Akt2 and MTSS1 Knockdown
2
3D Spheroid Invasion Assay in Collagen
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Briefly, the Friedl laboratory provided HT1080 cells. Before sharing among the two groups, the Sahai laboratory validated this cell line by comparing its STR profile to the published ones. Mycoplasma infection was excluded prior to the experiments. Cells were cultured in T75 flask with 10% CO2 at 37°C. 3D spheroid culture and labeling were performed as follows:
Multicellular spheroids containing 1,000 HT1080 cells were generated using hanging‐drop culture method (Del Duca et al, 2004 (link)).
The spheroids were embedded in rat‐tail collagen I (Corning, Cat no. 354249), in up to 18 wells of 96‐well imaging plates per collagen concentration, using 1 spheroid per gel and a final collagen concentration of 2.5 or 6 mg/ml. Former protocols for spheroid embedding (Wolf et al, 2013 (link); Haeger et al, 2014 (link)) were adapted to have control over the number of spheroids per well, spheroid height with respect to imaging window and the onset of collagen polymerization, to minimize variation between technical repeats per plate.
Plates were incubated for 24 h at 37°C to establish cancer cell invasion in three dimensions, prior to fixation in 4% PFA.
The 3D cell cultures were fluorescently stained with DAPI (Sigma, D9542, 2 μg/ml) and AlexaFluor633‐Phalloidin (Molecular Probes, A22284, 1:200 dilution) and stored (preferably for < 48 h) at 4°C prior to imaging.
3
Multiparametric Fluorescent Imaging Assay
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Tetramethylrhodamine, methyl ester (TMRM), Hoechst 33342, LysoTracker® Red DND-99, LysoTracker® Green DND-26, 5-(and-6)-chloromethyl-2′,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), CellLight® Nucleus-GFP, Alexa Fluor® 633 phalloidin, goat anti-mouse Alexa Fluor® 633, goat anti-rabbit Alexa Fluor® 633, goat anti-mouse Alexa Fluor® 488, and goat anti-rabbit Alexa Fluor® 488 were purchased from Molecular Probes (Thermo Fisher Scientific). Resazurin, pyrimethamine, methotrexate, aminopterin, and most of the drugs were purchased from Sigma. The rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark), mouse monoclonal anti-CHALV1 (Abcam, CSP, Cambridge, England), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-phospho-histone H2AX (γ-H2AX) (Millipore, Bedford, MA, USA), and rabbit polyclonal anti-cathepsin B (EMD/Calbiochem, San Diego, CA, USA) antibodies were purchased from each of the indicated companies.
4
Immunofluorescence Staining of H. pylori Infection
5
Antibody-Based Cytoskeletal Analysis
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Antibodies to acetylated tubulin and phospho-c-Raf-1 Y341 were from Sigma (St. Louis, MO). Antibodies directed to phospho-Erk1/2, Src, Fyn, Yes, Lyn, phospho-Src Y416, phospho-Pak2 Ser141 and phosphotyrosine were from Cell Signaling Technology (Danvers, MA). Antibodies to CXCR4 and ESM-1 were from R&D Systems (Minneapolis, MN). Alexa fluor® 633 phalloidin was from Molecular Probes (Eugene, OR). PP2 and PP3 were from Calbiochem (La Jolla, CA). Rat tail collagen type I was prepared as described previously [8 (link)].
6
Immunofluorescence Imaging of Transfected Cells
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A glass-bottom 24-well plate (MatTek Corporation , Ashland, MA) was coated with 1X poly-D-lysine (Sigma Aldrich), rinsed with water, and allowed to dry at room temperature. HEK-293A or ARPE-19 cells were plated at a density of 100,000 cells/well and transfected the following day with 500 ng of midi-prepped endotoxin-free plasmid DNA (Qiagen). Forty-eight hours after transfection, fresh media was added, and 24 h later (72 h post transfection), the cells were washed twice with 1X PBS (Fisher BioReagents, cat# BP2944100, Waltham, MA) followed by incubation with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) for 20 min. After PFA incubation, cells were washed with 1X PBS. For the ARPE-19 cells, the cell nuclei were stained with 300 nM 4’,6-diamidino-2-phenylindole (DAPI), dilactate solution (Molecular Probes, Eugene, OR). For membrane staining, the HEK-293A cells were washed twice with 1X PBS, fixed in 4% PFA, permeabilized in 0.1% Triton X-100 for 3 min, and washed again in 1X PBS. Cells were incubated in blocking buffer (1% bovine serum albumin [BSA] in PBS) for 10 min followed by Alexa Fluor™ 633 Phalloidin (1:50 dilution in PBS; Molecular Probes) for 20 min and washed twice with 1X PBS before being imaged using a 63X oil objective on a Leica SP8 confocal microscope (Buffalo Grove, IL).
7
Zebrafish Retinal Cell Immunohistochemistry
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Immunohistochemistry was perform as described [98 (link)], with the following antibodies: zpr-1 (cones; ZIRC), zpr-3 (rods; ZIRC), zrf-1/gfap (Muller glia cells; ZIRC), Zn8 (ganglion cells; ZIRC), and HuC/D (ganglion and amacrine cells; Molecular Probes). Embryos were cryosectioned at 12μm and incubated with primary antibodies diluted at 1:200 in block overnight at 4°C, then incubated with secondary antibody (anti-mouse Cy3) for 2hrs. Sections were counterstained with Alexa Fluor-633 Phalloidin at 1:100 and Sytox green at 1:10,000 (Molecular Probes) or mounted using Vectashield with DAPI (Vector Labs). Images were acquired using Zeiss LSM5 and/or Olympus FV1200 confocal microscopes, and analyzed using ImageJ with Cell Counter plug-in (imagej.nih.gov ).
8
Multimodal Cellular Characterization Assay
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Hoechst 33342 (H3570, 1:500), 2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA. C6827, 1:1000), ThiolTracker™ violet (T10095, 1:500), Alexa Fluor® 633 Phalloidin (A22284, 1:100), Goat anti-mouse Alexa Fluor® 488 (A11001, 1:500), Goat anti-rabbit Alexa Fluor® 633 (A21070, 1:500), Goat anti-mouse Alexa Fluor® 633 (A21050, 1:500) and Goat anti-rabbit Alexa Fluor® 488 (A11008, 1:500) were purchased from Molecular Probes (Invitrogen). Rabbit monoclonal anti-CD44 (EPR10133Y clone, ab51037, 1:1000) rabbit polyclonal anti-EpCAM (ab71916, 1:1000) and mouse monoclonal anti-CD90 (ab133350, 1:1000) were purchased from Abcam (CSP, Cambridge, England). Methotrexate, Doxorubicin, Cisplatin, Sorafenib, Sulfasalazine (SASP), Buthionine sulphoximine (BSO), Arsenic trioxide, Etoposide and Hydrogen peroxide (H2O2) were purchased from Sigma Chemicals. Mouse monoclonal anti-CD133/1 (AC133, 130-090-422, 1:100) was purchased from Miltenyi biotec (Bergisch Gladbach, Germany). Rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark, A000829. 1:500) and mouse monoclonal anti-β-actin (Sigma, A5441, 1:10,000) antibodies were purchased from each of the indicated companies.
9
Immunostaining Drosophila Egg Chambers
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After dissection, fly tissues were fixed with 3.8% paraformaldehyde in PBS (v/v) for 20 minutes at room temperature and washed 3 times with PBS. Fixed tissues were permeabilized with 0.03% Triton X-100 in PBS, (PBST) (v/v) for 1 hour, and stained with 250 ng/ml Hoechst 33342 (Molecular Probes, cat. no. H1399) and 0.3 μM F-actin (filamentous actin) stain Alexa Fluor 633 Phalloidin (Molecular Probes, cat. no. A22284) in PBST for 1 hour at room temperature, and washed 3 times with PBST every 5 minutes. To detect caspase activation by immunostaining, egg chambers were first incubated with the cleaved caspase-3 (Asp175) antibody (Cell signaling technology, 9661) diluted 1:200 in PBST with 1% bovine serum albumin (BSA) (v/v) at 4°C overnight, and then Alexa Fluor 633 Goat Anti-Rabbit IgG antibody (Molecular Probes, A-21070) diluted 1:200 in PBST with 1% BSA (v/v) at room temperature for 2 hours and washed 3 times with PBST after incubation of antibody. The stained tissues were mounted on glass coverslips with Vectashield mounting medium (Vector laboratories, H-1000). Images were captured with Zeiss LSM 780 confocal inverted microscope using a 20x, NA 0.8 Plan-Apochromat objective, and were analyzed using Zen 2013 or AxioVision 4.2 software (Carl Zeiss). Differential interference contrast (DIC) microscopy was used to image the morphology of tissues.
10
Immunohistochemistry and Immunofluorescence for COUP-TFII_V2
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IHC and IF with antibodies to COUP-TFII_V1 (cat. no. AB41859; RRID:AB_742211) and cytokeratin (CK)19 (cat. no. AB15463; RRID:AB_2281021; both from Abcam) were performed as previously described (18 (link),24 (link)). The custom-made primary antibody for COUP-TFII_V2 was used at 1:25 dilution in PBS containing 2% BSA (MilliporeSigma) and 0.01% Triton X-100 (Sigma). IHC for α-smooth muscle actin was performed with the monoclonal clone A4 (DAKO; Agilent Technologies, Inc.) diluted 1:100 in PBS with 2% BSA and 0.01% Triton X-100 following the previously described protocol; the sections were incubated with an M.O.M. kit (Vector Laboratories, Inc.) prior to incubation with the primary antibody. For β-catenin IF the same antibody used in western blot was used diluted 1:50; SMAD2/3P IF was performed with a 1:100 dilution of a rabbit polyclonal antibody (cat. no. ab272332; Abcam); actin filaments were decorated with Alexa Fluor 633 phalloidin (Molecular Probes cat. no. A22284); cell nuclei were stained with DAPI (cat. no. 10236276001; Roche). Collagen was stained with standard Sirius Red staining. Sirius Red was quantified with ImageJ 1.52r under Icy 2.0.3 on images with RGB color settings; the selection of stained areas was achieved with the function 'Color threshold'.
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