The procedure of immunofluorescence staining has been previously described [13 (link)]. Briefly, the attached cells on the gelatins were fixed for 5 min in 4% paraformaldehyde in PBS (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, followed by blocking in 1% bovine serum albumin (BSA) for 30 min. Subsequently, the cells were stained with 500 µL of 1% BSA containing 8 µL of anti-vinculin Alexa-fluor® 488 to detect vinculin (0.5 mg/ml; eBioscience, San Diego, CA, USA); 4 µL of Acti-stainTM 555 fluorescent phalloidin (14 µM; Cytoskeleton Inc., Denver, CO, USA) to detect F-actin; and 3 µL of 4’,6-diamidino-2-phenylindole (DAPI) solution (1 mg/mL; Dojindo Laboratories, Kumamoto, Japan) to detect the nuclei at 37 °C for 1 h, followed by incubation at 4 °C overnight. The specimen was then washed three times in PBS and once in deionized water. Finally, the specimen was mounted with ProLong® Diamond antifade mount reagent (Thermo Fisher Scientific Inc., Tokyo, Japan) and examined with a fluorescence microscope (Biorevo BZ-9000; Keyence Corp., Osaka, Japan).
Acti stain 555 fluorescent phalloidin
Manufactured by Cytoskeleton
Sourced in United States
Acti-stain™ 555 Fluorescent Phalloidin is a reagent used for the fluorescent labeling of F-actin in fixed and permeabilized cells. It is a high-affinity, stable probe that binds to F-actin and can be detected using a fluorescence microscope or flow cytometer.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Dojindo Laboratories (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Sp5 laser scanning confocal microscope, by Leica (1 mentions)
Y 27632, by Merck Group (1 mentions)
α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Sto 609, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Soluble mouse recombinant m csf, by R&D Systems (1 mentions)
Membrane dye dii, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
15 protocols using acti stain 555 fluorescent phalloidin
1
Immunofluorescence Staining Protocol for Analyzing Cell Cytoskeleton
2
Actin Cytoskeleton Analysis in Podocytes
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To identify changes in the organization of actin cytoskeleton, after the incubation of podocytes, cells were washed three times with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-X 100, incubated with 5% normal donkey serum (Sigma-Aldrich), then stained with Acti-stainTM555 Fluorescent Phalloidin for TRITC excitation filter setting (1:150 dilution; Cytoskeleton, Inc., CO, USA) and 4′,6-diamidino-2-phenylindole for the detection of actin cytoskeleton and nuclear DNA. Podocytes in permissive condition were observed as staining patterns and photographs were taken using fluorescent microscopy in a random manner, and the number of actin-stained filopodia per each cell were counted. Ten cells of each Tcf21-MP and Control-MP were analyzed, and the average number of filopodia between both cell lines was compared.
3
Immunofluorescent Staining of Cellular Structures
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Cells were initially seeded at 1x10 5 and grown for 5 days within the cloning rings. The cells were then fixed in 4% paraformaldehyde. 16HBE cells were immunofluorescently stained for the tight junction protein, zona-occludin-1 (ZO-1), using an anti-ZO-1 mouse IgG1antibody conjugated to Alexa 647 ® (Innova Bioscience, Cambridge, UK). The actin cytoskeleton of MRC5 cells was immunofluorescently stained using Acti-stain TM 555 fluorescent phalloidin (cytoskeleton, Inc, Denver, USA). Cell nuclei were visualised using DAPI contained in Prolong Gold mounting solution before being visualised by confocal microscopy (Leica SP5 laser scanning confocal microscope).
4
Endothelial Cell Contractility Regulation
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DPT was obtained from the Medicinal and Chemical Institute, China Pharmaceutical University. Y27632, taxol, compound C, STO-609 were purchased from Sigma-Aldrich (St. Louis, USA). Anti-CD31, RhoA, MLC, p-MLC (Thr18/Ser19), coffilin, p-coffilin (Ser3), AMPK, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), CaMKKβ, p-CaMKKβ (Thr286) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin and α-tubulin antibody were purchased from Santa Cruz Biotechnology (CA, USA). Acti-stain555 Fluorescent Phalloidin was purchased from Cytoskeleton (Denver, USA).
5
Osteoclast Differentiation Assay
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Sitagliptin was purchased from Meilun Biotech (Dalian, China) and dissolved in normal saline. Soluble mouse recombinant M-CSF and RANKL were obtained from R&D Systems (Minneapolis, MN, United States). Fetal bovine serum (FBS), alpha-MEM and penicillin were purchased from Gibco BRL (Gaithersburg, MD, United States). Triton X-100, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and TRAP staining solution were obtained from Sigma–Aldrich (St. Louis, MO, United States). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Kumamoto, Japan). The Acti-stain 555 fluorescent phalloidin was obtained from Cytoskeleton Inc. (Denver, CO, United States). The Cell Tracker Green, Fluo-4 AM, Hank’s balanced salt solution (HBSS), membrane dye DiI, and Pluronic F-127 were obtained from Life Technologies (Carlsbad, CA, United States). Primary antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay was purchased from Beyotime (Shanghai, China).
6
Immunocytochemical Analysis of Desmosomal Proteins
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Staining was performed, as previously described (6 (link)). The cells were fixed in 4% paraformaldehyde solution (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 15 min, following which they were incubated with donkey serum blocking buffer [Tris-buffered saline containing 5% (w/v) normal donkey serum (Jackson ImmunoResearch, Hamburg, Germany)] for 30 min and the primary antibody for 60 min. Subsequently, the cells were incubated with donkey anti-mouse polyclonal immunoglobulin G (IgG) and/or donkey anti-rabbit polyclonal IgG (DyLight 488 and 594, respectively; Jackson ImmunoResearch) for 60 min at room temperature. The following antibodies were used: Mouse monoclonal anti-DSC2 (7G6; Invitrogen Life Technologies), rabbit polyclonal anti-E-cadherin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-γ-catenin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-DSG2 (10G11; Progen Biotechnik GmbH), mouse monoclonal anti-PKP2 (PP2/62, PP2/86, PP2/150, Progen Biotechnik GmbH), mouse monoclonal anti-pan-cytokeratin (Santa Cruz Biotechnology, Inc.) and Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton Inc., Denver, CO, USA).
7
Cell Viability and Cytoskeletal Assessment
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On day 1 and day
14, cell-laden hydrogels were washed with Dulbecco’s PBS (DPBS)
for 5 min. Next, the cell-laden hydrogels were incubated with 0.3
μL/mL of Calcein-AM and 0.26 μL/mL of ethidium homodimer
for 1 h at room temperature protected from light. After 1 h, the cell-laden
hydrogels were washed three times for 5 min using DPBS. On day 14,
the cell-laden hydrogels were washed twice for 5 min with DPBS and
fixed with 4% paraformaldehyde for 45 min. Fixed cells within the
hydrogel were then stained with 140 nM of F-actin (Acti-stain 555
Fluorescent Phalloidin, Cytoskeleton, Inc.) in the presence of 1%
(v/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X100 at 4 °C
overnight. F-actin-stained cells in the hydrogel were washed three
times for 30 min with 1% (v/v) BSA and 0.3% (v/v) Triton X100, and
then counterstained with DAPI for 1 h at room temperature. The stained
cells in the hydrogel were washed with DPBS, and then imaged using
confocal microscopy (Olympus Fluoview, FV1000). For analysis, a total
of three hydrogels were imaged per condition with at least three random
images per gel.
14, cell-laden hydrogels were washed with Dulbecco’s PBS (DPBS)
for 5 min. Next, the cell-laden hydrogels were incubated with 0.3
μL/mL of Calcein-AM and 0.26 μL/mL of ethidium homodimer
for 1 h at room temperature protected from light. After 1 h, the cell-laden
hydrogels were washed three times for 5 min using DPBS. On day 14,
the cell-laden hydrogels were washed twice for 5 min with DPBS and
fixed with 4% paraformaldehyde for 45 min. Fixed cells within the
hydrogel were then stained with 140 nM of F-actin (Acti-stain 555
Fluorescent Phalloidin, Cytoskeleton, Inc.) in the presence of 1%
(v/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X100 at 4 °C
overnight. F-actin-stained cells in the hydrogel were washed three
times for 30 min with 1% (v/v) BSA and 0.3% (v/v) Triton X100, and
then counterstained with DAPI for 1 h at room temperature. The stained
cells in the hydrogel were washed with DPBS, and then imaged using
confocal microscopy (Olympus Fluoview, FV1000). For analysis, a total
of three hydrogels were imaged per condition with at least three random
images per gel.
8
Macrophage Morphology Observation Protocol
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Morphologies of treated macrophages were observed and photographed under an inverted microscope (ZEISS, German). For fluorescent observation, macrophages were fixed in paraformaldehyde and permeabilized beforehand. The cytoskeleton was labeled with Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton, USA) and nuclei with DAPI (YEASEN, China). Fluorescently labelled cells were examined using a ZEISS fluorescent imaging microscope (ZEISS, German).
9
Osteoclast Differentiation Assay
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DAC (CAS No. 31565-50-1, MOLBASE Biotechnology Co. Ltd, Shanghai, China) was dissolved in dimethyl sulfoxide without exposure to light and stored at −20°C. Dulbecco’s modified Eagle’s medium (DMEM) was provided by Hyclone (Logan, UT, USA). Penicillin–streptomycin solution, trypsin–ethylenediaminetetraacetic acid (EDTA) solution (0.25%), and fetal bovine serum (FBS) were obtained from Gibco (Gaithersburg, MD, USA). Recombinant mouse RANKL and macrophage-colony-stimulating factor (M-CSF) were provided by PeproTech (Rocky Hill, NJ, USA). A tartrate-resistant acid phosphatase (TRAP) staining kit, Triton X-100, and 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. A Cell Counting Kit-8 (CCK-8) was provided by Dojindo Molecular Technology Inc. (Kumamoto, Japan). Acti-stain 555 fluorescent phalloidin was obtained from Cyto-skeleton Inc. (Denver, CO, USA). The specific primary antibodies and secondary antibodies used in the experiments were provided by Cell Signaling Technology (Danvers, MA, USA).
10
Fibronectin-Coated Glass Coverslip Assay
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Glass coverslips were coated with 0.01% poly‐L‐lysine (P8920‐100ML, Sigma‐Aldrich), air‐dried overnight, and fixed with 0.5% glutaraldehyde. Fibronectin HiLyte 488 (FNR02‐A, Cytoskeleton, Denver, CO, USA) was added dropwise to each glass coverslip. The coverslips were then transferred to a 24‐well plate and incubated at 37 °C for 1 h. Cells were suspended following trypsinization and added to the glass coverslips. After 48 h, cells on each glass coverslip were fixed, permeabilized and labelled with Acti‐Stain™ 555 fluorescent phalloidin (PHDH1, Cytoskeleton), followed by mounting in anti‐fluorescence attenuating reagent (S2110, Solarbio, Beijing, China). For the determination of matrix degradation, the coverslips were photographed using a laser confocal microscope (ZEISS LSM 800, Carl Zeiss, Jena, Germany).
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