Coverslips were prepared as above mentioned with 0.2% porcine gelatin/PBS (Sigma-Aldrich) and cells were seeded 24 h after transfection. After another 24 h, the cells were fixed and stained for Cortactin (EMD Millipore; 1:1000) and Alexa Flour 488-conjugated phalloidin (Life Technologies; 1:50). For the analysis, 8 fields of view per coverslip from three biological replicates were imaged using a confocal microscope and analyzed for colocalization using the ImageJ software.
Alexa flour 488 conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated phalloidin is a fluorescent probe used to label and visualize actin filaments (F-actin) in cells. It binds tightly to F-actin, allowing for the detection and localization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Cortactin, by Merck Group (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Cultureslide, by BD (1 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (1 mentions)
Thapsigargin, by Cayman Chemical (1 mentions)
Fura 2 am, by Enzo Life Sciences (1 mentions)
U46619, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Rutin, by Cayman Chemical (1 mentions)
Eosin isothiocyanate, by Merck Group (1 mentions)
Oxidized glutathione gssg, by Merck Group (1 mentions)
Erp72, by Enzo Life Sciences (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti cofilin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti mouse igg and anti rabbit igg, by Merck Group (1 mentions)
12 protocols using alexa flour 488 conjugated phalloidin
1
Colocalization Analysis of Cortactin and F-Actin
2
Quantifying F-actin in HCT116 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
F-actin contents of HCT116 parental, WT and MUT cells were measured by Flow-Cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences San Jose, CA, USA). In brief, cells were cultured in a dish to 90% confluence, trypsinized and subsenquently divided into 2-ml microtubes (2×106 cells per tube), washed with PBS, fixed with 2% PFA for 1 h at room temperature, permeabilized with 0.1% Triton X-100 for 15 min and stained with Alexa Flour 488-conjugated phalloidin (A12379, Life Technologies) for 20 min at room temperature. After thorough washing with PBS containing 0.5% BSA, the cells were analyzed by flow cytometry and data analysis was carried out using Flow Jo (Tree Star Inc., Ashland, OR, USA).
3
Immunofluorescent Staining of Actin and Cortactin
4
Synthesis and Characterization of HPW-RX40
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HPW-RX40 (2-Methoxy-4-[(E)-2-nitrovinyl]phenyl 2,3-dichlorobenzoate) was synthesized according the methods described previously [17] (link). Bovine α-thrombin was purchased from Biovision, (Mountain View, CA, USA). Collagen (Type I, equine tendon) was from Helena Laboratories (Beaumont, TX, USA). Fibrinogen, U46619 (9,11-dideoxy-9,11-methanoepoxy PGF2α), 12-O-tetradecanoylphorbol-13-acetate (TPA), A23187, eosin isothiocyanate, oxidized glutathione (GSSG), and phenyl arsenoxide (PAO) were from Sigma-Aldrich (St. Louis, MO, USA). Alexa Flour 488-conjugated phalloidin, was obtained from Molecular Probes (Eugene, OR, USA). Thapsigargin and rutin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Human recombinant PDI, ERp72, and Fura-2/AM were purchased from Enzo Life Sciences (Farmingdale, NY, USA); Human recombinant ERp57 and anti-P47 Ab were from Abcam (Cambridge, UK), and ERp5 was from ProSpec Protein Specialists (Rehovot, Israel). Anti-PDI Ab was from Pierce/Thermo (Rockford, IL, USA); anti-GRP78 Ab was from Genetex (Irvine, CA, USA); phospho-PKC substrate antibody and anti-CHOP Ab were from Cell Signaling Technology (Beverly, MA, USA); anti-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma Chemical Co.
5
Berberine Chloride Protection Against Cisplatin Ototoxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the protective effects of berberine chloride against cisplatin-induced ototoxicity in the organ of Corti, we investigated morphological defects of IHCs and OHCs by staining stereocilia with phalloidin. At the end of the 30 h incubation period, all cochlear explants were washed with phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde in PBS. Permeabilization and staining was conducted simultaneously by Alexa Flour488 conjugated phalloidin (Invitrogen-Molecular Probes, USA) in 0.1% Triton X-100 in PBS for 1 h at room temperature (RT).
6
Cryosectioning and Staining of Mouse Duodenum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Duodenum from the small intestine of 2 month old C57/BL6 mice was harvested and immediately fixed and sub-dissected in 4% paraformaldehyde in PBS for 30 min. Tissue was then washed in PBS and cryoprotected overnight in 30% sucrose at 4 °C before embedding in OCT and freezing over an acetone-dry ice bath. Frozen blocks were stored at −80 °C until the time of sectioning. OCT blocks were sectioned using a Leica CM 1950 cryostat, at a thickness of 10 μm and melted on Superfrost Plus microscope slides (Fisher Scientific). All slides were kept at −80 °C until the time of staining. Slides were thawed and OCT washed out in room temperature PBS three times for 5 min each before staining with AlexaFlour488-conjugated Phalloidin (1:200, Molecular Probes) and DRAQ5 (1:200, ThermoScientific) for 2 hours. Subsequent to washing in PBS, samples were mounted with ProLong Gold antifade reagent (Molecular Probes) and #1.5 coverslip (Electron Microscopy Sciences).
7
Immunodetection of Cell Junctions
8
Immunoblotting Assay for Rac1 Activation
9
Visualizing Apoptotic Cell Phagocytosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine apoptotic thymocytes were induced by exposure to UV irradiation at 312 nm for 10 min. Transfected J774.1 cells treated with IFNγ and LPS and apoptotic cells stained with propidium iodide (Cell Signaling Technology) were co‐cultured (1:10) for 60 min and then extensively washed with phosphate‐buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, and blocked with 3% bovine serum albumin in PBS. Actin was stained with Alexa Flour 488‐conjugated phalloidin (Invitrogen Life Technologies). The coverslips were mounted using PermaFluor Mounting medium (Thermo Fisher Scientific, Miami, FL, USA). For confocal immunofluorescence analysis, cells were visualized with a Zeiss LSM 700 confocal microscope using Zen software.
10
Immunofluorescence Imaging of Endothelial Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HBMECs grown on collagen-coated coverslips in 24-well culture plates were fixed with 4% paraformaldehyde followed by blocking with donkey serum in 0.3 M glycine in PBS for 1 h at room temperature. The cells were then incubated with the following primary antibodies overnight at 4 °C: rabbit anti-VE-cadherin (1:200; Abcam), rabbit anti-occludin (1:200; Invitrogen) and rabbit anti-HA (1:500; Abcam). After rinses in PBS, cells were incubated with donkey secondary antibodies conjugated with DyLight 488 or Cy3 (1:1,000, Jackson ImmunoResearch Laboratories, Inc.). F-actin staining was done using Alexa Flour 488-conjugated phalloidin (1:2,000; Invitrogen). Cells were then counterstained with 4′,6-diamidino-2-phenylindole for nuclear labelling. After PBS rinses, coverslips were mounted on glass slides with antifade Vectashield solution (Vector Laboratories). Fluorescence images were captured with a confocal microscope. Immunofluorescence intensity of extracellular occludin and VE-cadherin was quantified by ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!