Proximal associations of mTOR with paxillin were detected using Duolink in situ PLA technology (Sigma-Aldrich) following the manufacturer’s instructions. Briefly, fixed and permeabilized HeLa cells were blocked with Duolink blocking solution and incubated with mTOR and paxillin antibodies overnight at 4°C in Duolink antibody diluent. As a negative control, an antibody against mitochondrial protein SDHA was used instead of mTOR. Cells were then washed and incubated with Duolink PLA probes (PLA probe anti-mouse Minus and PLA probe anti-rabbit Plus) in a preheated humidity chamber for 1 h at 37°C. Coverslips were incubated with the ligation solution, washed, and incubated with the amplification solution. Finally, coverslips were incubated with anti-mouse Alexa Fluor 488 secondary antibody to visualize paxillin and mounted on slides with Prolong Gold antifade reagent with DAPI (Thermo Fisher Scientific). PLA signals were detected with a Texas-Red filter and paxillin with a GFP filter on a DM5500 widefield fluorescence microscope (Leica Microsystems) using a 40× objective.
Dm5500 widefield fluorescence microscope
Manufactured by Leica
The Leica DM5500 is a widefield fluorescence microscope designed for laboratory applications. It offers high-quality optical performance and advanced imaging capabilities. The core function of the DM5500 is to enable users to visualize and analyze fluorescently labeled samples with precision and clarity.
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2 protocols using dm5500 widefield fluorescence microscope
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Proximal mTOR-Paxillin Interactions Detected
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Protein Interaction Visualization via Duolink
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Proximal associations of mTOR or LAMP1 with paxillin were detected using Duolink in situ PLA technology (Sigma-Aldrich), following the manufacturer's instructions. Briefly, fixed and permeabilized HeLa cells were blocked with Duolink blocking solution and incubated with mTOR or LAMP1 and paxillin antibodies overnight at 4°C in Duolink antibody diluent.
Negative-control coverslips were incubated with anti-paxillin antibody alone. Cells were then washed and incubated with Duolink PLA probes (PLA probe anti-mouse Minus and PLA probe anti-rabbit Plus) in a preheated humidity chamber for 1 h at 37°C. Coverslips were incubated with the ligation solution, washed and incubated with the amplification solution.
Finally, coverslips were mounted on slides with Prolong Gold antifade reagent with DAPI (Thermo Fisher Scientific). PLA signals were detected by a Texas-Red filter on a DM5500 widefield fluorescence microscope (Leica Microsystems) using a 40× objective.
Negative-control coverslips were incubated with anti-paxillin antibody alone. Cells were then washed and incubated with Duolink PLA probes (PLA probe anti-mouse Minus and PLA probe anti-rabbit Plus) in a preheated humidity chamber for 1 h at 37°C. Coverslips were incubated with the ligation solution, washed and incubated with the amplification solution.
Finally, coverslips were mounted on slides with Prolong Gold antifade reagent with DAPI (Thermo Fisher Scientific). PLA signals were detected by a Texas-Red filter on a DM5500 widefield fluorescence microscope (Leica Microsystems) using a 40× objective.
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