HCC cells were seeded on glass cover slips and incubated overnight for adhesion. Alexa Fluor™ 488 Phalloidin (Invitrogen, #A12379 ) staining was performed as previously described for adhesion. Alexa Fluor™ 488 Phalloidin (Invitrogen, #A12379 ) staining was performed as previously described48 (link). Imaging was performed at IBG Optic Imaging Core Facility with fluorescent microscope (Olympus—BX61).
Alexa fluor 488 phalloidin
Manufactured by Thermo Fisher Scientific
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Alexa Fluor 488 phalloidin is a fluorescent dye that selectively binds to F-actin, a component of the cytoskeleton. It is used in microscopy and flow cytometry applications to visualize and study the distribution and organization of actin filaments within cells.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (190 mentions)
Triton x 100, by Merck Group (96 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (88 mentions)
Hoechst 33342, by Thermo Fisher Scientific (83 mentions)
Bovine serum albumin (bsa), by Merck Group (56 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (45 mentions)
Lsm 710, by Zeiss (45 mentions)
Paraformaldehyde (pfa), by Merck Group (39 mentions)
Lsm 700, by Zeiss (34 mentions)
Lsm 510, by Zeiss (23 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (23 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (22 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (21 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (20 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (20 mentions)
Lsm 700 confocal microscope, by Zeiss (17 mentions)
Lsm 880, by Zeiss (16 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (16 mentions)
Vectashield, by Vector Laboratories (16 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (16 mentions)
1 398 protocols using alexa fluor 488 phalloidin
1
Actin Cytoskeleton Visualization in HCC Cells
2
F-actin Polymerization in Neonatal PMN
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F-actin polymerization assay was performed using Alexa Fluor 488 phalloidin (Invitrogen), as described previously (37 (link)). In brief, 5 x 105 neonatal PMN were co-incubated with C. parvum or plain medium for the negative controls for 3 h at 37°C and 5% CO2 in 12 x 75 mm polypropylene tubes (BD Falcon). Then, the cells were fixed with Fixation/Permeabilization® solution (BD Biosciences, San Diego, CA, USA) and stored at 4°C until stained with Alexa Fluor 488 phalloidin (Invitrogen). First, an aliquot of 100 μl was centrifuged at 600 x g for 10 min at room temperature (RT). Then, the supernatant was discarded, and the sample was suspended in 100 μl of Alexa Fluor 488 phalloidin (1:1,000 in PBS with 1% BSA). After an incubation of 30 min at RT in the dark, the samples were centrifuged at 600 x g for 10 min, the supernatant discarded, and the samples were washed with 300 μl of sterile PBS before centrifuging once more at 600 x g for 10 min at RT. Finally, the cells were suspended in 400 μl of sterile PBS, and the acquisition was performed in the FL1 (FITC) channel in a BD Accuri C6 plus® flow cytometer. Analysis was performed in FlowJo v 10.6.2 software over at least 10,000 events in the active gate.
3
Retinal Pigment Epithelium Flat Mount
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After enucleation, mouse eyes were punctured with a fine needle and immersion fixed in ice-cold 10% neutral-buffered formalin (NBF) for 10 minutes. The anterior segment was removed, and the neural retina was separated from the RPE-choroid-sclera. Six radial cuts were made toward the optic nerve to flatten the posterior eyecup. After total fixation of 30 minutes in NBF, the tissue was washed three times with PBS, incubated with PBST (PBS with 0.1% Tween-20) for 20 minutes, and blocked with 5% goat serum in PBST for 30 minutes. For phalloidin staining, tissues were incubated with Alexa Fluor 488-phalloidin (1:300; Invitrogen) in blocking solution overnight at 4°C; for double staining, tissues were incubated with ZO-1 antibody (1:300 dilution) in blocking solution overnight at 4°C, washed with PBST, and incubated with Alexa Fluor 555–conjugated goat anti-rabbit antibody (1:200; Invitrogen) and Alexa Fluor 488 phalloidin (1:300; Invitrogen) for 2 hours. Nuclei were stained by incubating with 4′,6-diamidino-2-phenylindole (DAPI) for 20 minutes. The whole RPE-choroid-sclera tissue was then flat mounted, with RPE side up, onto slides and cover-slipped for imaging. Fluorescent images were captured with a Leica SP5 confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA), and identical imaging parameters were applied to both WT and mutant RPE flat mounts.
4
Vesicle-Induced Actin Cytoskeleton Analysis
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In order to investigate the effects of exposure to vesicles obtained from size exclusion chromatography on the actin cytoskeleton, Alexa Fluor 488® Phalloidin (Molecular Probes®) was used as a fluorescent dye according to manufactures instructions. Briefly, after 24 and 48 h treatment, cells were fixed in 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100 and stained with 3 U/mL Alexa Fluor 488® Phalloidin for 30 min as cell nuclei were counterstained with Hoechst 33342 (Molecular Probes®). Experiments were evaluated with a fluorescence microscope ImageXpress Micro High Content System (Molecular Devices). Nine sites from each experiment were analyzed in triplicate using MetaXpress software (Molecular Devices).
5
Dual Fluorescent Staining of Actin and Cilia
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For phalloidin staining of fibrillar actin and anti-acetylated tubulin staining of cilia, the embryos were fixed in 4% PFA/PTwTx (1× PBS, 0.1% Tween 20, 0.2% Triton X100) for 1 h at room temperature, washed 5 times with PTwTx, incubated in 100% acetone pre-cooled to -20 °C for 7 min on ice, and washed 3 more times with PTwTx. Then, the embryos were incubated for 2 h in blocking solution (95% v/v of 1% BSA/PTwTx and 5% v/v of heat-inactivated sheep serum). Blocked embryos were stained overnight at 4 °C in 0.4U of Alexa Fluor 488 Phalloidin (ThermoFisher) and 0.1 µl of mouse monoclonal anti-acetylated tubulin (Sigma) dissolved in 100 µl blocking solution. Unbound primary antibody and phalloidin were washed away by five 10 min PTwTx washes, and the embryos were stained for 2 h at room temperature in the dark in 0.4U of Alexa Fluor 488 Phalloidin and 0.1 µl of Alexa Fluor 568 rabbit anti-mouse IgG (Molecular Probes) dissolved in 100 µl blocking solution. After five more 10 min PTwTx washes, the embryos were gradually embedded in Vectashield (Vectorlabs) and imaged with the Leica SP8 CLSM.
6
Fluorescent Cytoskeleton Reconstitution
7
Visualizing F-actin in Fibroblast Hydrogels
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We labeled the F-actin in fibroblasts with Alexa Fluor™ 488 Phalloidin (A12379, Thermo Fisher Scientific). In brief, fibroblast-embedded hydrogels were fixed with 4% paraformaldehyde/PBS at 4 °C overnight, followed by triple PBS washes. The samples were permeabilized with 0.1% Trion X-100 for 30 min, followed by triple PBS washes. The samples were blocked in 1% bovine serum albumin for 1 h at room temperature and then incubated in Alexa Fluor™ 488 Phalloidin for 1 h. Subsequently, the samples were washed with PBS and stained with 1 μg/ml DAPI solution (62248, Thermo Fisher Scientific). The fluorescence images were captured using a Zeiss LSM 880 confocal microscope.
8
Visualizing Cellular Components in C2C12 Cells
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C2C12 cells grown on glass coverslips were stained according to the manufacturer’s instructions. The endoplasmic reticulum was stained using l g/mL of Alexa Fluor®, 647 Concanavalin A Conjugates (Thermo Fisher Cat# A12379) for 1 h. The cytoskeleton was stained using l g/mL of Alexa FluorTM 488 Phalloidin (Thermo Fisher Cat# C21421) for 30 min. Nuclei were labeled using DAPI for 15 min. Images were acquired on a ZEISS fluorescence microscope using a 20× or 40× objective.
9
Fluorescence In Situ Hybridization and Immunostaining
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The conditions and methods for fluorescence in situ hybridization and fluorescence in situ hybridization combined with immunostaining are described in detail by Lécuyer, E. et al.12 (link),15 (link),28 (link). The following antibodies were used for fluorescence in situ hybridization: Biotin-SP (long spacer) IgG fraction monoclonal mouse anti-Digoxin (1:400; Jackson ImmunoResearch Laboratories Inc.; 200-062-156), Peroxidase IgG fraction monoclonal mouse anti-Biotin (1:100; Jackson ImmunoResearch Laboratories Inc.; 200-032-211), TSA Fluorescein (1:50; PerkinElmer; FP1013), and TSA Reagent Alexa Fluor 594 Tyramide (1:50; Molecular Probes; T20950). The following antibodies were used for immunostaining: rabbit polyclonal anti-anillin IgG (1:500; Provided by Dr. Maria G. Giansanti) (Giansati et al., 2015; Sechi et al., 2017), mouse anti-dlg IgG (1:100; Developmental Studies Hybridoma Bank (DSHB); 4F3), rabbit polyclonal anti-RFP IgG (1:100; MBL; PM005), mouse monoclonal anti-Lamin IgG (1:100; DSHB; ADL67.10) and mouse monoclonal anti-Peanut IgG (1:100; DSHB; 4C9H4). Actin was stained with Alexa FluorTM 488 Phalloidin (1:100; Thermo Fisher Scientific; A12379). Samples were inspected with a confocal laser-scanning microscope (Olympus FLUOVIEW FV10i).
10
Larval Muscle Integrity Analysis
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Pupae were prepared for scanning electron microscopy by sputter coating with Au90Pd10 and imaged with in a FEI-XL30 Field Emission Gun Environmental Scanning Electron Microscope. Larvae were dissected in low Ca2+ saline, HL-3 (Pesavento et al. 1994 (link)) in a magnetic chamber (Budnik et al. 2006 ), and fixed in 4% paraformaldehyde in PBS for 1 h. F-actin was labeled with Alexa FluorTM 488 Phalloidin (ThermoFisher). All 30 muscles in 8 hemisegments (A2–A5) were scored each larva (240 muscles per animal) for signs of reduced integrity (torn, thin, loss of sarcomeric structure, or missing).
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