For fluorescence microscopy, cells were fixed in 4% paraformaldehyde (50-980-487, Fisher Scientific) in PBS for 15 min. Then, they were permeabilized using 0.2% Triton X-100 (Sigma–Aldrich) in PBS for 20 min at room temperature. The actin cytoskeleton was stained with ActinRed (2 drops/mL of PBS; R37112, Invitrogen) for 30 min protected from light at room temperature. Similarly, the DNA was stained with 1:1000 diluted Hoechst 33342 (16.2 mM; Invitrogen) for 10 min protected from light at room temperature. Fluorescent images were taken using a fluorescence microscope (BZ-X800, Keyence).
Actinred
Manufactured by Thermo Fisher Scientific
Sourced in United States
ActinRed is a fluorescent probe designed to visualize and quantify the F-actin cytoskeleton in cells. It binds selectively to F-actin and can be used for live-cell or fixed-cell imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Nucblue, by Thermo Fisher Scientific (5 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Bz x800, by Keyence (2 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (2 mentions)
Fixation permeabilization kit, by BD (2 mentions)
Alexafluor 488 secondary goat anti mouse antibody, by BioLegend (2 mentions)
Actingreen, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Lsm 780, by Olympus (1 mentions)
R37112, by Thermo Fisher Scientific (1 mentions)
Stage top incubator, by Tokai Hit (1 mentions)
Pkh26, by Merck Group (1 mentions)
Pkh67, by Merck Group (1 mentions)
Elyra ps 1, by Zeiss (1 mentions)
780 upright confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
18 protocols using actinred
1
Fluorescence Microscopy of Cytoskeleton and DNA
2
Immunofluorescence Analysis of Epithelial-Immune Cell Interactions
3
Imaging and Quantifying F-Actin Dynamics
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All microscope imaging was performed with a Zeiss LSM 780 or Olympus SD-OSR. To assess the F-actin organization after certain treatments, cells were seeded on a glass-bottom culture dish (D11130H, Matsunami). After culture with the peptides, cells were fixed with 4% paraformaldehyde phosphate buffer solution (PFA, 30525-89-4, Wako) for 10 min at room temperature, washed, and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 15 min. Cells were washed with DPBS twice and incubated with ActinRed (Rhodamine-conjugated phalloidin, R37112, Invitrogen) or ActinGreen (Alexa-Fluor-488-conjugated phalloidin, R37110, Invitrogen) for 15 min at room temperature. Cells were washed with DPBS before being imaged. Morphological characteristics, including cell spreading area and perimeter area ratio, were quantified by Image J.
For live-cell time-lapse imaging, transfected HuH-7 cells were placed inside a stage-top incubator (Tokai Hit) and were tracked with a 100x/1.35 Silicon UPlanSApo objective for more than 12 h. 5.5-µm stack images were acquired every 20 min, and imaging parameters were adjusted to minimize photobleaching and avoid cell death.
For live-cell time-lapse imaging, transfected HuH-7 cells were placed inside a stage-top incubator (Tokai Hit) and were tracked with a 100x/1.35 Silicon UPlanSApo objective for more than 12 h. 5.5-µm stack images were acquired every 20 min, and imaging parameters were adjusted to minimize photobleaching and avoid cell death.
4
Internalization of Small Extracellular Vesicles
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The separated sEVs were labeled in vitro for 4 min using the green fluorescent dye PKH67 (Sigma-Aldrich, MO), after which complete medium was added to prevent overstaining; then, ultracentrifugation was carried out for an additional 70 min at 120,000 × g and 4 °C. After washing with PBS, sEVs were added to HK‐2 cells and incubated for 24 h. PBS was used to wash the cells twice. ActinRed (Invitrogen, USA) was used to stain the cytoskeleton. The nuclei were counterstained with DAPI. In vivo, the sEVs were labeled with PKH26 (Sigma-Aldrich, MO) according to the same steps described above21 (link). Laser scanning confocal microscopy was used to assess the internalization of sEVs both in vitro and in vivo.
5
Immunofluorescent Staining of Epithelial-Immune Cell Coculture
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Epithelial monolayers on the apical side of Transwell membranes and macrophages/dendritic cells on the basolateral side were washed with fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FBS) and stained with primary mouse anti-human CD14 (BD Pharmingen, catalog no. 347490) according to previously established protocols (8 (link)). Following a wash with FACS buffer, cells were fixed and permeabilized with a Fixation/Permeabilization kit (BD Biosciences, catalog no. 554714) and concurrently stained with an Alexa Fluor 488 secondary goat anti-mouse antibody (BioLegend, catalog no. 405319), NucBlue (Invitrogen, catalog no. 12303553), and ActinRed (Invitrogen, catalog no. 15119325). The membranes were mounted on glass slides with Prolong Diamond Antifade (Invitrogen, catalog no. 15205739) and cured for 24 hours. Images were acquired with the Zeiss LSM 880 confocal microscope at a 63× magnification and curated with the Zeiss ZEN software.
6
Immunofluorescence Microscopy of Flavivirus Infection
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For immunofluorescence microscopy, C6/36 persistently infected cells were seeded on round coverslips and fixed with 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.2, for 20 min. The samples were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Pre-incubation was performed with 50 mM ammonium chloride and 3% BSA in PBS, pH 8.0, for 45 min to block the free aldehyde groups. The samples were then incubated with a primary anti-flavivirus antibody 4G2 hybridoma, gently provided by Prof. Luciana Arruda and Prof. Fábio Gomes (UFRJ) at a 1:100 dilution for 1 h, rinsed, and incubated with a 1:400 dilution of the secondary goat anti-human IgG antibody conjugated to AlexaFluor 488 (Invitrogen) at room temperature for 1 h. Actin was stained with actin red (Invitrogen) for 20 min in the dark. After rinsing with PBS and mounting with prolong antifade (Vector Labs, Burlingame, CA, USA), the slides were visualized using a Zeiss Elyra PS.1, using Confocal mode.
7
CFSE Labeling and Uptake of P.gingivalis
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P.gingivalis was labeled with 10 μM carboxyfluoresceine succinimidyl ester (CFSE ebioscience, Invitrogen, Thermofisher Scientific West Columbia, SC, USA) for 1 hour at 37 degree with shaking, then residual stain removed by successive washings and bacterium suspended at 10 MOI for uptake experiments, with/without Cytochalasin D 1uM (Sigma Aldrich, St Louise, MO, USA) for 48 hours, after which cells were harvested and analyzed by flow cytometry and SA-B-gal staining. Cells were harvested, thoroughly washed and then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained with actin ActinRed 555 ReadyProbes Reagent and nuclear counterstaining using DAPI mounting medium; ProLong Gold Antifade Mountant (Invitrogen, Thermofisher scientific, Waltham MA, USA), then images were taken using Zeiss 780 upright confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
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P.gingivalis was labeled with 10 μM carboxyfluoresceine succinimidyl ester (CFSE ebioscience, Invitrogen, Thermofisher Scientific West Columbia, SC, USA) for 1 hour at 37 degree with shaking, then residual stain removed by successive washings and bacterium suspended at 10 MOI for uptake experiments, with/without Cytochalasin D 1uM (Sigma Aldrich, St Louise, MO, USA) for 48 hours, after which cells were harvested and analyzed by flow cytometry and SA-B-gal staining. Cells were harvested, thoroughly washed and then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained with actin ActinRed 555 ReadyProbes Reagent and nuclear counterstaining using DAPI mounting medium; ProLong Gold Antifade Mountant (Invitrogen, Thermofisher scientific, Waltham MA, USA), then images were taken using Zeiss 780 upright confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
8
Fluorescence Microscopy of Cytoskeleton and DNA
9
Cellular Permeabilization and Immunostaining
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After fixation, cells were permeabilized with 0.1% Triton X-100 solution for 10 min at room temperature. Cells were incubated with Nucblue (nuclei, Thermo fisher) and actinRed (Thermo fisher) or actinGreen (Thermo fisher).
Immunocytochemistry imaging data was collected using a Zeiss LSM 880 confocal microscope at 10× magnification. Images for data analysis after blood perfusion were obtained using an EVOS M5000 imaging system (Thermo Fisher) at 4× magnification.
Immunocytochemistry imaging data was collected using a Zeiss LSM 880 confocal microscope at 10× magnification. Images for data analysis after blood perfusion were obtained using an EVOS M5000 imaging system (Thermo Fisher) at 4× magnification.
10
Endothelial Integrity Assessment
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After overnight incubation, the endothelial layer integrity was assessed using three conditions: healthy endothelium (control), endothelium with embedded macrophages (sample) and TNF-α treated endothelium (inflamed). Inflamed samples were treated with 5 ng/ml TNF-α (Peprotech) for 4 hours. Cells were fixated and cells were permeabilized with 0.1% Triton X-100 solution for 10 min at room temperature. Cells were incubated with Nucblue (nuclei, Thermo fisher) and actinRed (Thermo fisher). Immunocytochemistry imaging data was collected using a Zeiss LSM 880 confocal microscope at 10× magnification.
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