Cells or tissue sections were permeabilized with 0.1% Triton X‐100 for 10 min, blocked with goat serum for 30 min, and incubated with HMGB1 (1:100 dilution, T55060, Abmart, China), TOMM20 (1:100 dilution, CL594‐66777, Proteintech, China), or F‐actin (1:200 dilution, C2201S, Beyotime, China) antibodies for 16–18 h at 4 °C. The next morning, the sections were incubated for 1 h at room temperature with ATF594‐coupled secondary antibody (direct label antibodies do not require secondary antibody staining). Cell nuclei were re‐stained with DAPI. Fluorescence images were acquired by a confocal fluorescence microscopy.
F actin
Manufactured by Beyotime
Sourced in China
F-actin is an essential structural protein found in the cytoskeleton of eukaryotic cells. It plays a crucial role in various cellular processes, including cell motility, cell division, and maintaining cell shape. F-actin is a key component of the actin filaments, which are dynamic and versatile structures that undergo constant assembly and disassembly.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Alexa fluor 647 goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica Microsystems (1 mentions)
Phalloidin fitc, by Beyotime (1 mentions)
Fv1200, by Olympus (1 mentions)
Goat anti rabbit igg as1109, by Aspen (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Affinity Biosciences (1 mentions)
Collagen type 1, by Proteintech (1 mentions)
Cd45 pe 30 f11, by Thermo Fisher Scientific (1 mentions)
Snail, by Affinity Biosciences (1 mentions)
Pd l1, by Proteintech (1 mentions)
7 protocols using f actin
1
Immunofluorescence Staining of HMGB1, TOMM20, and F-actin
2
Integrin β3 and Focal Adhesion Proteins
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The cells on fibrin network were fixed with 4% paraformaldehyde (Beyotime, China) and permeated with 0.2% TritonX‐100 (Sigma‐Aldrich, USA). Cells were blocked with 1% BSA in PBS and incubated with Integrin β3 (Affinity, AF6086, 1:100), Zyxin (Abcam, ab109316, 1:500), c‐Src (Proteintech, 60315‐1‐Ig, 1:100), and CCR7 (Abcam, ab32527, 1:100) antibody overnight at 4 °C. After incubation, the cells were washed and incubated with Alexa Fluor 647 goat anti‐rabbit secondary antibody (Beyotime, China) and Alexa Fluor 594 goat anti‐mouse secondary antibody (Emarbio, China) at 20 °C for 1 h. Then the cells were washed and stained for F‐actin (Beyotime, China) and nuclei with DAPI staining solution (Beyotime, China). Fluorescent images were observed with a confocal laser scanning microscope (FV3000 Olympus, Japan).
3
Visualizing V. parahaemolyticus Infection in HeLa Cells
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According to a previous report (Tandhavanant et al., 2018 (link)), assays of infection of HeLa cells were conducted. HeLa cells were plated in 24-well dishes at a density of 1.5 × 105 per cell, grown for 12 h, and then infected with WT, ΔvmeL, and CΔvmeL V. parahaemolyticus at MOI of 10 for 2 h. After 2 h f co-incubation, the cells were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. 0.5% Triton-X was used to permeabilize the cells for 10 minat room temperature. Then, the cells were probed with rhodamine-phalloidin (SBS Genetech, Shanghai, China) to stain F-actin and DAPI (Beyotime, Shanghai, China) to highlight HeLa cell DNA. Images were captured using an inverted fluorescence microscope (Axio observer Z1, ZEISS).
4
Exosome Uptake Visualization in Macrophages
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For the exosome uptake experiment, exosomes were labelled with a PKH67 Fluorescent Cell Ligation kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. The exosomes were then washed with 0.5% BSA/PBS (cat. no. P0007; Beyotime Institute of Biotechnology) to remove excess dye. The labelled exosomes were again ultracentrifuged at 100,000 x g for 1 h at 4°C to remove residual dye; then, the exosomes (at a concentration of 10 µg/ml) were incubated with macrophages at 37°C for 8 h in the dark. The cell nuclei and cytoskeleton were separately stained with DAPI (cat. no. C1005; Beyotime Institute of Biotechnology) for 5 min at room temperature and F-actin (cat. no. KA4118; AmyJet Scientific) for 30 min at room temperature and then examined by laser scanning confocal microscopy (magnification, ×800).
5
Cellular Immunofluorescence Staining Protocol
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For cellular immunofluorescence staining, cells were attached to cell slides in 24-well plates. For F-actin staining, the cells were grown on collagen gels for 24 h. Then the cell slides and collagen were fixed in 4% PFA for 20 min at room temperature and permeabilized with 0.1% Triton X-100 for 15 min. Then incubated with primary antibody against E-cadherin (1:1000; CST), vimentin (1:500; Santa Cruz Biotechnology), F-actin (1:100, Beyotime) overnight at 4 °C. Nucleus were stained with 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). Fluorescence images were visualized using a fluorescence microscope (Leica Microsystems, Mannheim, Germany).
6
Immunofluorescence Staining of CXCL12
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Frozen tissue sections and cells grown on glass-coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, and blocked in 10% normal goat serum for 1 h. The anti-CXCL12/SDF1 rabbit polyclonal antibody (Cat#: AF6612, Beyotime Biotechnology, 1:100) was diluted in blocking buffer and placed on the cells at 4°C overnight. After the incubation, slides were washed three times in PBS and then incubated with goat anti-rabbit IgG (AS1109; Aspen Biotechnology, Wuhan, China; 1:100) for 1 h at 37 °C. For filamentous actin (F-actin) staining, cells on coverslips were incubated with FITC-phalloidin (Cat#: C1033, actin-tracker green, Beyotime Biotechnology) in PBS for 2 h at room temperature. Nuclei were stained using 4′6′-diamidino-2-phenylindole (DAPI) solution for 10 min at room temperature. Imaging was performed using an FV1200 (Olympus Corporation) confocal microscope.
7
Comprehensive Protein Expression Analysis
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The antibodies of Smad2/3 (3102, CST), AKT (#9272, CST), p-AKT (#9271, CST), p-Smad2/3 (#ab272332, Abcam), E-cadherin (#20874-1-AP, Proteintech), N-cadherin (#22018-1-AP, Proteintech), Vimentin (#BF8006, Affinity Biosciences), Collagen Type I (#14695-1-AP, Proteintech), NF-κB1 (#14220-1-AP, Proteintech), LEF-1 (#28540-1-AP, Proteintech), PD-L1 (#66248-1-Ig, Proteintech), GAPDH (#60004-1-Ig, Proteintech), SNAIL (#AF6032, Affinity Biosciences), ROCK1 (#PTM-6454, PTM Bio), ROCK2 (#PTM-6169, PTM Bio), Ki67 (#GB111499, Servicebio), and fibrillar actin (F-actin) (#C1033, Beyotime) were used for western blot, immunofluorescence staining, and immunohistochemical staining. The following antihuman antibodies were used for flow cytometry: CD45 (PE, 30-F11; eBioscience), CD8α (APC, 53-6.7; eBioscience), 7-AAD (Invitrogen), CD3 (eFluor™ 506, UCHT1; eBioscience), PD-1 (Pacific Blue, EH12.2H7; Biolegend), and TIM-3 (FITC, F38-2E2; Biolegend).
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