NIH 3T3 fibroblasts were obtained from ATCC (Manassas, VA) and maintained in DMEM (HyClone, Logan, UT) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 incubator at 37°C. Primary antibodies against phospho-AktS473, phospho-MekS217/221, Pan-Mek, phospho-S6 ribosomal protein235/236, phosphop-70S6KT421/S424, phospho-ERK1/242/44, phospho-4EBP37/46 and pan-Akt were purchased from Cell Signaling (Boston, MA). Primary antibodies against phospho-p70S6KSer411 were purchased from Abcam (Cambridge, MA). Bleomycin and primary antibodies against β-actin and fibronectin were purchased from Sigma (St. Louis, MO). Anti-collagen-I was purchased from Rockland (Gilbertsville, PA). All HRP-conjugated secondary antibodies were obtained from Bio-Rad (Hercules, CA) and Alexa-labelled secondary antibodies were purchased from Life Technologies (Carlsbad, CA). TGFβ was purchased from R&D (Minneapolis, MN), whereas LY294002 inhibitor, U0126 inhibitor, and Rapamycin were purchased from EMD Millipore Bioscience (San Diego, CA). PF4708671 inhibitor was obtained from Santa Cruz Biotechnology (Dallas, TX). Adenoviral particles encoding CA-Mek1 and DN-Mek1 were obtained from Cell Biolabs, Inc. (San Diego, CA), whereas retroviral plasmids encoding CA-Akt1 and DN-Akt1 were generated in the laboratory [6 (link)].
Alexa labelled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
The Alexa-labelled secondary antibody is a fluorescent labeling reagent used in immunofluorescence assays. It binds to the primary antibody and emits a fluorescent signal upon excitation, enabling the detection and visualization of target proteins or molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Harmony software, by PerkinElmer (5 mentions)
Operetta high content imaging system, by PerkinElmer (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Bleomycin, by Merck Group (1 mentions)
Phospho s6 ribosomal protein235 236, by Cell Signaling Technology (1 mentions)
Phospho p70 s6k t421 s424, by Cell Signaling Technology (1 mentions)
Anti collagen 1, by Rockland Immunochemicals (1 mentions)
Nih 3t3 fibroblasts, by American Type Culture Collection (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti vp16, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Anti ps2, by Cell Signaling Technology (1 mentions)
Anti ps1, by Cell Signaling Technology (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Completetm protease inhibitor cocktail, by Roche (1 mentions)
Trizol reagent, by Roche (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
9 protocols using alexa labelled secondary antibody
1
Intracellular Signaling Pathway Analyses
2
Reagents and Antibodies for Protein Analysis
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Reagents for Western blotting and BCA protein assay kit were from Pierce (Rockford, IL, USA), membranes and ECL+ from GE Healthcare (Little Chalfont, UK). Primary antibodies anti‐PS1 and anti‐PS2 were from Cell Signaling (Danvers, MA, USA), anti‐VP16 and anti‐Notch1 were from Abcam (Cambridge, UK), and anti‐actin and anti‐tubulin antibodies and the fluorescent nucleic acid stain DAPI were obtained from Sigma‐Aldrich (St Louis, MO, USA). Primary antibody directed against APP‐CTF was kindly received from N. Sergeant (INSERM, Lille, France). Secondary antibodies coupled to peroxidase were obtained from Amersham Bioscience (Uppsala, Sweden). The applied dilutions for each antibody were according to the manufacturer's instructions. Alexa‐labelled secondary antibodies were obtained from Life Technologies. Fluorescent mounting medium was from DAKO (Agilent Technologies, Santa Clara, CA, USA). TRIzol reagent and CompleteTM protease inhibitor cocktail were from Roche (Basel, Switzerland). The cDNA synthesis kit and iQ SYBR Green Supermix were from Bio‐Rad (Hercules, CA, USA).
3
Quantification of γH2AX foci in EA.hy926 cells
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EA.hy926 EC were grown on glass coverslips in 6-well plates for 48 h, treated with TNF-α, NAC or were mock-treated and irradiated as described before. At 1 h, 4 h and 24 h post irradiation cells were fixed with 3.7% paraformaldehyde (15 min, AppliChem, Darmstadt, Germany) at room temperature (RT), and permeabilization was performed by addition of 0.25% Triton-X 100 in PBS for 15 min, followed by blocking in 3% bovine serum albumin (BSA) in PBS for 60 min. Next, EA.hy926 cells were incubated with anti-phospho-histone H2AX specific (γH2AX, 1:1000, Millipore, Darmstadt, Germany) and anti-centromere protein F (CENP-F) primary antibodies (1:2000, Santa Cruz, Heidelberg, Germany) to discriminate cells in G1 and S/G2 cell cycle phases [24 (link)] followed by appropriate Alexa-labelled secondary antibodies (Invitrogen, Darmstadt, Germany). Subsequently, nuclei were counterstained with DAPI solution (Invitrogen) and coverslips were mounted with Vectashield (Vector Laboratories, Peterborough, UK). Images were taken using an AxioImager Z1 microscope, equipped with an Axiocam camera and Axiovision 4.6 software (Zeiss, Göttingen, Germany). For quantification of γH2AX foci formation 40 G1 and 40 S/G2 phase cells as differentiated by CENP-F signal intensity were evaluated per experiment. At least three independent experiments were performed for each data point.
4
Immunofluorescence Labeling Protocol
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The following antibodies were used: rabbit-αRab11 (715300. Invitrogen), mouse-αTfR (10R-CD71aHU. Fitzgerald, Acton, MA), mouse-αHA (MMS-101P. Covance, Princeton, NJ), chicken-αGAPDH (GW22763. Sigma-Aldrich, Saint Louis, MO), Alexa-labelled secondary antibodies (Invitrogen), IRdye-labelled secondary antibodies (Li-Cor, Lincoln, NE). The fluorophores used for protein conjugation were: SeTau-647 (SeTa Biomedicalos, Urbana, IL), CF-488 and CF-594 (Biotium, Hayward, CA). Alexa-594-labelled WGA was from Thermo Fischer (Grand Island, NY). Nocodazole was from Sigma-Aldrich.
5
Immunofluorescence Staining of Cultured Cells
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Following compound treatment, cells were fixed in 3.7% paraformaldehyde in PBS at room temperature for 15 minutes, washed with PBS, blocked with 5% normal goat serum in 0.3% Triton X100 in PBS for 1 hour at room temperature then incubated with primary antibody diluted in antibody dilution buffer (1% BSA, 0.3% Triton X100 in PBS) at 4 °C for 16 hours. Cells were washed with PBS then incubated with an Alexa-labelled secondary antibody (1:500, Life Technologies) and Hoechst 33342 (1 μg/ml) in antibody dilution buffer at room temperature for 60 minutes. Following washing with PBS, cells were imaged with an Operetta high content imaging system (Perkin Elmer) at 10× or 20× magnification and analysed using Harmony software (Perkin Elmer).
6
Immunofluorescence imaging of cells
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Following compound treatment, cells were fixed in 3.7% paraformaldehyde in PBS at room temperature for 15 min, washed with PBS, blocked with 5% normal goat serum in 0.3% Triton X100 in PBS for 1 h at room temperature then incubated with primary antibody diluted in antibody dilution buffer (1% BSA, 0.3% Triton X100 in PBS) at 4°C for 16 h. Cells were washed with PBS then incubated with an Alexa‐labelled secondary antibody (1:500, Life Technologies) and Hoechst 33342 (1 μg/ml) in antibody dilution buffer at room temperature for 60 min. Following washing with PBS, cells were imaged with an Operetta high content imaging system (Perkin Elmer) at 10× or 20× magnification and analysed using Harmony software (Perkin Elmer).
7
Single Cell Immunofluorescent Imaging Protocol
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Single cell immunofluorescent imaging was conducted as previously described36 (link). In brief, following compound treatment, cells were fixed in 3.7% paraformaldehyde in PBS at room temperature for 15 minutes, washed with PBS, blocked with 5% normal goat serum in 0.3% Triton X100 in PBS for 60 minutes at room temperature then incubated with primary antibody diluted in antibody dilution buffer (1% BSA, 0.3% Triton X100 in PBS) at 4 °C for 16 hours. Cells were washed with PBS then incubated with an Alexa-labelled secondary antibody (1:500, Life Technologies) and Hoechst 33342 (1 μg/ml) in antibody dilution buffer at room temperature for 60 minutes. Following washing with PBS, cells were imaged with an Operetta high content imaging system (Perkin Elmer) at 10× or 20× magnification and analysed using Harmony software (Perkin Elmer).
8
Immunofluorescent Imaging Protocol
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Following compound treatment, cells were fixed in 3.7 % paraformaldehyde (in PBS) at room temperature for 15 minutes, washed with PBS, blocked with 5% normal goat serum in 0.3 % Triton X100 in PBS for 1 hour at room temperature and then incubated with primary antibody diluted in antibody dilution buffer (1 % BSA, 0.3 % Triton X100 in PBS) at 4°C for 16 hours. Cells were washed with PBS then incubated with an Alexa-labelled secondary antibody (1:500, Life Technologies, Inchinnan, UK) and Hoechst 33342 (1 μg/ml) in antibody dilution buffer at room temperature for 60 minutes. Following washing with PBS, cells were imaged with an Operetta high content imaging system (Perkin Elmer, Sear Green, UK) at 10x or 20x magnifications and analyzed using Harmony software (Perkin Elmer). Typically 3 to 5 fields per well were imaged which equated to between approximately 1000 and 4000 cells/well.
9
High-Content Cell Cycle Analysis
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Determination of cell cycle fractions was conducted using high content imaging as previously described.24 For multiparametric cell cycle analysis, cells were labelled with 10 μM EdU for 15 min immediately prior to fixation with 3.7% paraformaldehyde in PBS at room temperature for 15 min. Cells were washed twice in PBS then twice in 3% BSA in PBS before permeabilization with 0.5% Triton X100 in PBS for 20 min at room temperature. Cells were washed twice with 3% BSA in PBS before incorporated EdU was labelled with an Alexa Click‐iT EdU labelling kit (Life Technologies). Following blocking for 30 min with 5% normal goat serum in PBS, cells were incubated with an anti‐pHH3 (S10) primary antibody diluted in antibody dilution buffer (1% BSA, 0.3% Triton X100 in PBS) at 4°C for 16 h. Cells were washed with PBS then incubated with an Alexa‐labelled secondary antibody (1:500, Life Technologies) and Hoechst 33342 (1 μg/ml) in antibody dilution buffer at room temperature for 60 min. Following washing with PBS, cells were imaged with an Operetta high content imaging system (Perkin Elmer) at 10× magnification and analysed using Harmony software (Perkin Elmer). The antibodies used are listed in Table S1 .
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