Cells cultured on confocal dishes were fixed with 4% PFA for 20 min at room temperature, permeabilized and incubated with primary antibodies at 4 °C. All primary antibodies were used at a 1:200 dilution. For in situ proximity ligation assay, protein–protein interactions between CD36 (Thermo) and Integrin β1 (abcam) were detected with secondary proximity probes (Anti-Rabbit Plus and Anti-Mouse Minus) according to the Duolink In Situ Fluorescence protocol (Sigma-Aldrich).
Duolink in situ fluorescence protocol
Manufactured by Merck Group
Duolink® In Situ Fluorescence Protocol is a laboratory equipment product. It enables the detection and analysis of protein-protein interactions in fixed cells or tissue samples using a fluorescence-based method.
Automatically generated - may contain errors
Lab products found in correlation
Integrin β1, by Abcam (1 mentions)
Anti rabbit plus, by Merck Group (1 mentions)
Anti mouse minus, by Merck Group (1 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (1 mentions)
Detection reagents red kit, by Merck Group (1 mentions)
Anti myc tag 9b11, by Cell Signaling Technology (1 mentions)
Dm6000 fluorescent microscope, by Leica (1 mentions)
5 protocols using duolink in situ fluorescence protocol
1
Protein Interaction Detection in Cells
2
Interactome Analysis of FGFR1 Signaling
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To analyze the interactions between FGFR1.GF, FGFR1 and their protein partners, Duolink® In Situ Fluorescence Protocol was used (Sigma-Aldrich). U2OS-SBP-R1.GF cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton in PBS. Cells were then incubated with appropriate antibodies and treated according to the manufacturer’s protocols. Cell nuclei were stained with NucBlue Live dye.
3
Duolink in situ Fluorescence Assay
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PLA was performed according to the Duolink in situ Fluorescence protocol (Sigma). Briefly, cells were grown on glass coverslips, fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 in PBS for 20 min at room temperature. Cells were blocked for 1 h at 37 °C with Duolink Blocking Solution and then the blocking solution was replaced with the primary antibodies at 4 °C overnight. The subsequent step was incubating cells with the Duolink in situ PLA Probe Anti-Rabbit PLUS and Anti-Mouse MINUS PLA probes (Sigma) for 1 h at 37 °C. Then, cells were incubated with ligation solution for 30 min at 37 °C and amplification solution for 100 min at 37 °C. Finally, coverslips were mounted onto slides with Duolink in situ Mounting Medium with DAPI (Sigma).
4
Duolink Fluorescent Protein Interaction
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To analyze the interaction between studied proteins, Duolink® In Situ Fluorescence Protocol was used (Sigma-Aldrich). U2OS-FGF12-GFP.myc cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton in PBS. Cells were then incubated with appropriate antibodies and treated according to the manufacturer’s protocols. Cell nuclei were stained with NucBlue Live dye. Cytoplasm was stained with HCS Cell Mask Deep Red dye.
5
Immunofluorescence Staining and Microscopy
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Immunofluorescence staining was conducted as described previously (30 (link)). Primary antibodies used were mouse monoclonal anti-MYC-Tag 9B11 (1:750; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-HA Y-11 (1:100; Santa Cruz, Dallas, TX), mouse monoclonal anti-HA 16B12 (1:100; BioLegend, San Diego, CA), rabbit monoclonal anti-Na+/K+/ATPase (Alexa Fluor® 488), EP1845Y (1:50; Abcam, Cambridge, United Kingdom), and rabbit monoclonal anti-Na+/K+/ATPase EP1845Y (1:250; Abcam).
A Zeiss LSM 510 confocal microscope with × 40 objective was used to perform confocal microscopy (Carl Zeiss AG, Oberkochen, Germany). Epifluorescent microscopy was performed using × 40 objective on a Leica DM6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The Duolink In Situ Fluorescence Protocol with Detection Reagents Red Kit (Sigma) was used as per the manufacturer's instructions. Cells were transfected for 48 hours before fixation, permeabilization, and addition of anti-MYC and anti-HA antibodies.
A Zeiss LSM 510 confocal microscope with × 40 objective was used to perform confocal microscopy (Carl Zeiss AG, Oberkochen, Germany). Epifluorescent microscopy was performed using × 40 objective on a Leica DM6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The Duolink In Situ Fluorescence Protocol with Detection Reagents Red Kit (Sigma) was used as per the manufacturer's instructions. Cells were transfected for 48 hours before fixation, permeabilization, and addition of anti-MYC and anti-HA antibodies.
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