Immunocytochemical staining was used to visualize the DSBs and distinguish between the cell cycle stages G1 and S/G2. Four hours before fixation, a nucleoside analogue of thymidine, 5-ethynyl-2′-deoxyuridine (EdU; Thermo Fisher Scientific), was added at a final concentration of 10 μM. The cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton-X (both from Sigma-Aldrich, St. Louis, MO, USA). Overnight incubation with primary antibodies against γH2AX (Biolegend, San Diego, CA, USA) and 53BP1 (Santa Cruz Biotechnology, Dallas, TX, USA) was followed by 1 h of incubation with a secondary antibody conjugated with Alexa 555 (Cell Signaling Technology, Danvers, MA, USA). The samples were stained with the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific) to visualize EdU according to the manufacturer’s instructions. Finally, the nuclei were stained with Hoechst dye (BisBenzemide H33342; 1 μg/ml; Sigma-Aldrich).
Alexa 555
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa 555 is a fluorescent dye that can be used for labeling and detection of biomolecules in various biological applications. It has an excitation maximum of 555 nm and an emission maximum of 565 nm, making it suitable for visualization and quantification purposes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Cell Signaling Technology (3 mentions)
5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions)
γh2ax, by BioLegend (1 mentions)
Triton x, by Merck Group (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Tra 1 81, by Cell Signaling Technology (1 mentions)
Antibodies directed against cd14, by Miltenyi Biotec (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti vinculin clone hvin 1, by Merck Group (1 mentions)
Macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Marienfeld (1 mentions)
Plan apochromat 63x 1.40 na, by Zeiss (1 mentions)
Anti human ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
6 protocols using alexa 555
1
Visualizing DNA Double-Strand Breaks in Cell Cycle
2
Immunostaining of Induced Pluripotent Stem Cells
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Cells were fixed in 4% paraformaldehyde in PBS and immunostained using standard protocols. Antibodies against Nanog (Rabbit mAb, Cat.No. 4903s, Cell Signaling) and TRA-1-81 (Mouse mAb, No. 4745s, Cell Signaling) were used to verify the pluripotency of the iPSCs after gene editing. The antibodies used to identify the neurons derived from the iPSCs were neuron-specific class III β-tubulin (TuJ, Rabbit mAb, Cat.AB15708, Millipore) and Microtubule-associated protein 2 (Map 2, Mouse mAb, Cat. AB34918, Millipore). The cells were incubated at 4°C overnight in the primary antibodies, which were diluted with 10% goat serum and 0.3% Triton X-100 in PBS. The dilutions used were 1:800 (Nanog), 1:1000 (TRA-1-81), 1:400 (Map), and 1:200 (TuJ1). The cells were then treated with a secondary antibody (goat-anti mouse Alexa 555, 1:200, Cell Signaling Technology, Cat. #4409 and/or Alexa Fluor® 594 goat-anti rabbit Alexa 488, 1:200, Cell Signaling Technology, Cat. #4412) diluted in the same solution with the primary antibodies for 60 minutes at room temperature. The cells were then stained with a DAPI solution (diluted in PBS with the rate 1:1000) for 10 minutes. Between these steps, the cells were rinsed three times with 0.5% Tween in PBS.
3
Isolation and Differentiation of Human Monocytes
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Human monocytes were isolated from blood of healthy donors as described previously (Van Goethem et al., 2010 (link)). Cells were re-suspended in cold PBS supplemented with 2 mM EDTA, 0.5% heat-inactivated fetal calf serum (FCS) at pH 7.4 and magnetically sorted with magnetic microbeads coated with antibodies directed against CD14 (Miltenyi Biotec). Monocytes were then seeded on glass coverslips at 1.5x106 cells/well in six-well plates in RPMI 1640 (Invitrogen) without FCS. After 2 h at 37°C in humidified 5% CO2 atmosphere, the medium was replaced by RPMI containing 10% FCS and 20 ng/mL of Macrophage Colony-Stimulating Factor (M-CSF) (Peprotech). For experiments, cells were used after seven days of differentiation.
Macrophages plated on glass coverslips (0.17 mm ±0.005mm, Marienfeld) were unroofed and fixed as previously described (Bouissou et al., 2017 (link)) and labelled with Alexa Fluor 488-phalloidin (Molecular Probes, 1/500) and Alexa Fluor 647-coupled phalloidin (Molecular Probes, A22287, 1/100) for RIM and dSTORM, respectively. Vinculin was stained using mouse anti-vinculin (clone hvin-1, Sigma-Aldrich, 1/500) and a secondary F(ab')2 coupled to Alexa 555 (Cell Signaling technology).
Macrophages plated on glass coverslips (0.17 mm ±0.005mm, Marienfeld) were unroofed and fixed as previously described (Bouissou et al., 2017 (link)) and labelled with Alexa Fluor 488-phalloidin (Molecular Probes, 1/500) and Alexa Fluor 647-coupled phalloidin (Molecular Probes, A22287, 1/100) for RIM and dSTORM, respectively. Vinculin was stained using mouse anti-vinculin (clone hvin-1, Sigma-Aldrich, 1/500) and a secondary F(ab')2 coupled to Alexa 555 (Cell Signaling technology).
4
Imaging Protein Localization in Cancer Cells
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Peptidase M84 treated (2.0 μg/ml for 18 h) and untreated PA-1 and SKOV3 cells were fixed with 4% paraformaldehyde for 10 min at RT. Cells were then permeabilized with 0.1 % Triton X-100 in 0.1% sodium citrate solution and blocked with 5% FBS. Cells were incubated O/N with primary antibody (1:200) against the protein of interest (as per requirement) at 4°C in a moist chamber. Cells were washed with PBS and incubated with either Alexa 488 (Cell Signaling Technology Cat# 4408 and Cell Signaling Technology Cat# 4412) or Alexa 555 conjugated secondary antibody (Cell Signaling Technology Cat# 4413 and Cell Signaling Technology Cat# 4409) for 2 h at RT. Nuclei were stained with either DAPI or Hoechst 33342 (working concentration 1.0 μg/ml) for 10 min at 37°C. Cells were washed twice and mounting was done with 10% glycerol. Images were captured using confocal microscope (Zeiss 710); objective-Plan-Apochromat 63X / 1.40 NA. c and all the other required analysis was done according to the procedure described by Das et al., 2022.83 (link)
5
Immunofluorescence and Immunohistochemistry Protocols
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Studies were performed as described previously [16] (link), [17] (link), [19] (link). The primary antibody for MELK (1∶200, Sigma-Aldrich, Missouri) was used to visualize the fluorescent signals using the following secondary antibodies: Alexa 488 or Alexa 555 (1∶1000, Cell Signaling Technology, MA). Specificity was determined using no-primary control slides. For immunohistochemistry, the following primary antibodies were used: Nestin (anti-Nestin, clone 10C2, 1∶200, mouse monoclonal antibody, MAB5326, MA) and Ki67 (anti-Human Ki-67, clone MIB-1, 1∶1, mouse monoclonal antibody, Dako, Denmark). The Envision system (Dako) followed by Diaminobenzidine (DAB) method was used for detection of primary antibody according the manufacturer’s protocol. For paraffin-embedded slides, hematoxylin was used as a nuclear counterstain. IHC scoring was performed using automated digital image analysis (ImageJ).
6
Subcellular Localization of F Protein Mutants
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Cells were grown on 24-mm coverslips and transfected with plasmids expressing YLMY motif-mutated F protein and AP1M1–AP5M1. After 24 h, the cells were harvested and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at 4 °C. Subsequently, the fixed cells were permeabilized using 0.2% Triton X-100 in 0.1% sodium citrate and blocked with 5% bovine serum albumin. The cells were then incubated with anti-Myc or anti-Flag antibody to detect AP and F protein, respectively, followed by an incubation with a secondary antibody conjugated to Alexa 488 or Alexa 555 (Cell Signaling Technology), and finally counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) to label the nuclei. Fluorescence signals were captured with a Nikon A1 confocal microscope (Nikon, Tokyo, Japan). The Pearson’s coefficients were calculated by analysing each cell separately in confocal sections.
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