MIA PaCa-2 cells were seeded at 10,000 cells per well in 8-well chambered coverslides (Nunc Lab-Tek, #155383, Waltham, MA, USA), treated or not with doxycycline at 2 µg/mL, and allowed to attach for 48 h. Cells were washed with PBS, fixed in 4% paraformaldehyde (ProSciTech, #C006) and permeabilized with 0.1% Triton X-100 (MERCK, X-100, Darmstadt, Germany) for 10 min at room temperature each and stained using ActinGreen 488 Ready Probes Reagent (Invitrogen, #R37110, Waltham, MA, USA) according to the manufacturer’s protocol. Cells were imaged using the Leica Digital LightSheet confocal microscope TCS SP8 DLS.
Actingreen 488 readyprobes reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ActinGreen™ 488 ReadyProbes™ Reagent is a fluorescent labeling solution designed for the detection of F-actin in fixed and permeabilized cells. The reagent contains a green-fluorescent dye that selectively binds to F-actin, allowing for the visualization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Lsm 780, by Zeiss (4 mentions)
Fluorsavetm reagent, by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Nucblue fixed cell readyprobes reagent, by Thermo Fisher Scientific (3 mentions)
Tcs sp5, by Leica camera (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Axiocam icc5, by Zeiss (2 mentions)
Hoechst, by Enzo Life Sciences (2 mentions)
Axio vert a1 fluorescence microscope, by Zeiss (2 mentions)
Nucblue fixed cell stain readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Hcx pl apo 1.25 n a, by Leica camera (2 mentions)
Murine egf, by Thermo Fisher Scientific (2 mentions)
His tag, by Thermo Fisher Scientific (2 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (2 mentions)
55 protocols using actingreen 488 readyprobes reagent
1
Visualizing Actin Cytoskeleton in MIA PaCa-2 Cells
2
Immunolocalization of Heat Shock Proteins
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Immunolocalization of HSP 90, 70, 60 and 27 was performed in all hemocyte cultures (controls, F24 and F48). The cells were fixed in 4% Paraformaldehyde (Sigma-Aldrich; PFA) in Phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) in PBS. The cells were incubated overnight at 4°C with primary antibodies all from Enzo Life Sciences: HSP90α/β monoclonal antibody, HSP70 monoclonal antibody, HSP60 (insect) polyclonal antibody or HSP27 polyclonal antibody. Antibodies were diluted 1:40 in PBS with 1% bovine serum albumin (BSA, Sigma-Aldrich). The cells were then incubated in 4% BSA-PBS for two hours to prevent non-specific antibody binding. Following this, the hemocytes were incubated for a further two hours at room temperature with secondary antibody DyLight 488, Goat Anti-Mouse IgG (Abbkine) for HSP 90 and 70 or Dylight 594, Goat Anti-Rabbit IgG (Abbkine) for HSP 60 and 27. Concentrations of secondary antibodies were 2μg/ml. ActinGreen 488 ReadyProbes Reagent (Invitrogen) or ActinRed 555 ReadyProbes Reagent (Invitrogen) were used to label the actin fibers. The cell nuclei were stained with Hoechst (Enzo Life Sciences). Fluorescence signals were analyzed by fluorescent microscopy an Axio Vert.A1 fluorescence microscope (Zeiss) with Axio Cam ICc 5 (Zeiss).
3
Quantitative Immunofluorescence Assay for Cell Differentiation
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Differentiation was confirmed morphologically by permeabilizing with 0.17 mM Triton X-100 (Fisher Scientific) for 5 minutes, then fixing in 4% buffered paraformaldehyde (Fisher Scientific) and staining with 4',6-diamidino-2-phenylindole (DAPI) (NucBlue Fixed Cell Stain ReadyProbes reagent) for 5 minutes at stock concentration and phalloidin-conjugated Alexa Fluor 488 (ActinGreen 488 ReadyProbes reagent) (both from Invitrogen, Carlsbad, CA, USA) for 30 minutes also at stock concentration according to the manufacturer's protocols. Cells were imaged with an Olympus microscope (model BX34F) with an attached Olympus DP73 digital color camera and Olympus U-HGLGPS fluorescent light source (Olympus, Shinjuku, Tokyo, Japan). The percentage of differentiated cells (kidney-shaped nucleus) was calculated to be 69%, comparable to the percentage reported in literature (see Supplemental Section 1 ) [47 (link), 53 (link)].
4
Caco-2 Cells Permeabilization and Staining
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Caco-2 cells were permeabilized with 0.03% Triton X-100 (Sigma, T8787) in PBS for 10 min and washed twice with 4% FBS in PBS solution. Actin and nuclear stainings were performed using the direct stains ActinGreen 488 ReadyProbes Reagent (Invitrogen, R37110) and NucBlue Fixed Cell ReadyProbes Reagent (Invitrogen, R37606). Direct staining was performed following the manufacturer’s instructions and under constant flow.
5
Cellular Uptake of Nanoparticles by Macrophages
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To evaluate the cellular uptake of nanoparticles by macrophages, cells were seeded at 2.5 × 104 cells/cm2 and treated the day after with Nano and KP-Nano suspended at 1 μg/mL in the completed medium. After incubation at 37 °C for 2 or 4 h, the cells were washed, fixed with 4 vol% paraformaldehyde in PBS at room temperature, and treated with ActinGreen™ 488 ReadyProbes™ Reagent (Invitrogen™, Carlsbad, CA, USA) to stain the cytoskeleton and with Hoechst 33342 Solution (Thermo Scientific™, Waltham, MA, USA) to stain the nuclei. Images were acquired by a Nikon A-1 confocal microscope and analyzed by the use of NIS-Elements software.
6
Immunocytochemistry for NRP1 and β-Actin
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C3H10T1/2 cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 3 min on ice. After blocking with 5% bovine serum albumin (BSA) in PBS for 2 h, the cells were incubated with corresponding antibodies. For localization of NRP1 and β-actin, the cells were incubated with NRP1 antibody (Abcam; 1:250) overnight at 4 °C, and then incubated with Alexa Fluor 594–conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) (1:300), followed by staining with ActinGreen™ 488 ReadyProbes™ Reagent (Invitrogen). The stained cells were counterstained with DAPI and were observed using a confocal laser scanning microscope LSM780 (Carl Zeiss, Oberkochen, Germany) at Central Research Laboratory, Okayama University Medical School. The procedure of colocalization of NRP1 and JIP4 with immunocytochemistry was shown in Supplementary Materials and Methods .
7
Fluorescent Visualization of Cytoskeletal Dynamics
8
Visualizing F-actin in Cultured Cells
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Forty-eight hours after seeding of cells on Petri dishes, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton ×100 for 5 min. Samples were washed with PBS after each step and then incubated with ActinGreen 488 ReadyProbes Reagent (Invitrogen, ThermoFisher, Waltham, MA, USA, R37110) (2 drops in 1 mL PBS) for F-actin staining for 30 min at room temperature. Finally, cells were stored in PBS at 4 °C prior to image acquisition. Nikon Eclipse Ti inverted epi-fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) with 40× objective lens was used to observe cells and collect fluorescent images.
9
Immunofluorescence Analysis of SDC4 in HUVECs
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Young and senescent HUVECs were seeded in EGM-2 media at a density of 1 × 10 4 cells/well on poly-D-lysine coated slides. Cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 1 h at 4 °C. Cells were washed again in PBS and blocked with 5% BSA for 1 h at room temperature, followed by incubation with SDC4 antibody (5G9) (sc-12766, Santa Cruz Biotechnology, USA) in 1% BSA overnight at 4 °C and with secondary anti-mouse Alexa Fluor 568 antibody (cat. no. A20184, Invitrogen), at room temperature in 1% BSA for 1 h. The actin filaments were labelled with ActinGreen ™ 488 ReadyProbes ™ Reagent (R37110, Invitrogen). Cells were stained with nuclear HOECHST 33342 (cat. no. H-3570; Molecular Probes, Oregon, USA) in PBS for 5 min. Finally, cells were cover slipped with Vectashield mounting media (H-1200, Vector Laboratories, Burlingame, CA) and viewed with fluorescence microscopy (Nikon Eclipse 80i, Nikon, Japan). Omission of the primary antibody resulted in a lack of labeling, confirming the specificity of the antibody. The fluorescence intensity of SDC4 staining was quantified in at least 100 cells for each condition and each replicate using the CellProfiler image analysis software, version 4.2.0 [23] .
10
Fluorescent Cytoskeleton Imaging Protocol
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Phalloidin staining was carried out to label and identify F-actin (cytoskeleton) expression in the cells to observe the cellular morphology. On day 7, the samples were fixed with 4% paraformaldehyde for 20 min and washed with PBS. The samples were then permeabilized with 0.1% TritonX-100 for 30 min and 0.1% Tween-20 for 15 min. Blocking buffer was prepared with 0.1%Tween-20, 2% bovine serum albumin, and 2% goat serum. Phalloidin (Invitrogen™ ActinGreen™ 488 ReadyProbes™ Reagent) was used in the concentration of 2 drops/mL of blocking buffer and incubated for 1 h. After incubation, the samples were washed with PBS and stained with Hoechst (Invitrogen™ Hoechst 33342, Eugene, OR, USA) (1:1000 concentration Hoechst: PBS) for 5 min. The samples were then imaged using fluorescence microscopy (EVOS™ FL Auto 2 imaging system, ThermoFisher, Waltham, MA, USA).
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