Rabbit MT-SP1 (Calbiochem) antibody was used for immunostaining and western blot (WB) of THP1 cells. HGEC were treated with 1 μM of human recombinant MSP1 (R&D Systems) with or without RON inhibitor (200 nM) for varying times. WB analysis was performed with rabbit anti-p44/p42 MAPK (Erk1/2), rabbit anti-phospho-p44/p42 Erk1/2, rabbit anti-pan-Akt, and rabbit anti-phospho-Akt antibodies (all from Cell Signaling Technology). Mouse anti-β-actin antibodies and phalloidin-FITC conjugate were obtained from Sigma-Aldrich.
Phalloidin fitc conjugate
Manufactured by Merck Group
Sourced in United States, Spain
Phalloidin-FITC conjugate is a fluorescent probe used for the detection and visualization of F-actin in cells. It binds specifically to F-actin, allowing for the labeling and microscopic imaging of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546 donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Hoechst 33256, by Merck Group (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Rabbit anti pan akt, by Cell Signaling Technology (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Mouse monoclonal antibodies, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti nmiia antibody, by BioLegend (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Anhydrous pyridine, by Merck Group (1 mentions)
Potassium persulfate, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
α amylase, by Merck Group (1 mentions)
Amyloglucosidase, by Merck Group (1 mentions)
Lipase, by Merck Group (1 mentions)
Dmem and ham s f12, by Thermo Fisher Scientific (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Vimentin v9, by Santa Cruz Biotechnology (1 mentions)
A1r laser scanning confocal microscope, by Nikon (1 mentions)
5 protocols using phalloidin fitc conjugate
1
MSP1-Induced Erk1/2 and Akt Signaling
2
Immunofluorescence Analysis of Cell Markers
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After treatments, cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde in PBS at 25 °C for 20 min and permeabilized with 0.1% Triton-X100 in PBS at 25 °C for 20 min. Fixed cells were incubated with 3% BSA in PBS at 25aC for 1h and further incubated for 24 h with different mouse monoclonal antibodies (Santa Cruz Biotechnology): β-catenin (1:50), E-cadherin (1:100), vimentin V9 (1:100), ZO-1 (1:50). After labeling, samples were washed with PBS and incubated with a mixture of Alexa Fluor® 546 donkey anti-mouse IgG 1:250 (Invitrogen-Thermo Fisher), phalloidin-FITC conjugate 1:50 (Sigma-Aldrich) and 2.5 μM Hoechst 33256 (Sigma-Aldrich) for 60 min. Then, samples were washed with PBS and mounted with a drop of Vectashield Mounting Medium (Vector Laboratories). Samples were examined with a Nikon Eclipse Ti microscope (with an objective Plan apo VC 60x, 1.4 DIC ½) with acquisition software Micrometric SE Premium (Accu-Scope). A minimum of 10 fields containing several cells were collected from each sample. All images were processed using Fiji ImageJ 1.52b Software (National Institutes of Health, USA).
3
Immunofluorescence Detection of NMIIA in A431 Cells
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To detect the NMIIA isoform in A431 cell line, cells were seeded into glass coverslip-containing 24-well plates. Cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. Samples were blocked with 2% w/v Bovine Serum Albumine solution, endogenous NMIIA was detected using anti-NMIIA antibody (rabbit, polyclonal, 1:300, Biolegend) and Alexa Fluor-546-conjugated anti-rabbit secondary antibody (Life Technologies). Actin filaments were stained by phalloidin-FITC conjugate (Sigma-Aldrich). Immunofluorescence was imaged using a Zeiss Axio Observer Z1 microscope equipped with 40x EC Plan-Neofluar objective, Colibri illumination system and AxioCam MRm camera.
4
Simulated Gastrointestinal Digestion of Compounds
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2,2′-Azinobis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS), porcine bile extract, (FBS), gallic acid, 6-hydroxy-2,5,7,8-tetramethyl- 2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), porcine pancreas pancreatin, cellulose membrane dialysis tubing (12,000 Da molecular weight cut-off), Hoechst 33256, phalloidin-FITC conjugate, N,Obis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + TMS), pyridine anhydrous (99.8%), potassium persulfate, and the enzymes used in the simulated gastrointestinal digestion (α-amylase (EC 3.2.1.1), amyloglucosidase (EC 3.2.1.3), lipase (EC 3.1.1.3), and pepsin (E.C 3.4.23.1)) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). Ferric trichloride (FeCl3), iron (II) sulfate (FeSO4), sodium chloride (NaCl), potassium oxalate, paraformaldehyde, triton X-100, and sodium acetate were obtained from Panreac Química, S.L.U. (Barcelona, Spain). DMEM and Ham's F12, foetal bovine serum (FBS), antibiotic mixture, trypsin-EDTA were purchased from GIBCO®. Mouse monoclonal antibodies for β-catenin, E-cadherin, vimentin V9 and ZO-1 were obtained from Santa Cruz Biotechnology. Vectashield Mounting Medium was purchased from Vector Laboratories. Alexa Fluor® 546 Donkey anti-mouse IgG was obtained from Invitrogen-Thermo Fisher.
5
Visualizing Cytoskeletal Structure and Cell Nuclei
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Cells were fixed with freshly prepared paraformaldehyde (3.7% in PBS) and permeabilized with Triton X-100 (0.5% in PBS) for 15 min at room temperature. Phalloidin-FITC conjugate (Sigma Aldrich, Madrid, Spain), which binds polymerized F-actin, was used to identify cytoskeletal actin filaments, while DAPI (4′, 6-diamidino-2-phenylindole) was used to mark the DNA and to recognize cell nuclei. All samples were mounted with antifade medium Vectashield (VectorLabs, Barcelona, Spain) and were examined with a Nikon A1R confocal scanning laser microscope. For the analysis of cells grown on the HFs, the samples were inverted and a series of optical sections were obtained using the objective Plan Apo 20X DIC N2 equipped with a 405 nm argon laser and 561 nm HeNe laser to detect cell nuclei (DAPI) and cytoskeleton (Phalloidin-FITC), respectively. The reconstructions of 37 images (15 μm step−1) of confocal sections were assembled using NIS Elements 3.2 software (Boston, Massachusetts, USA).
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