In situ PLA was performed as described in the previous report [17 (link)]. Briefly, cells were seeded on a sterile 12-mm coverslip, transfected with full-length GFP-CPAP, GFP-CPAP fragments or Myc-STAT3 fragments, and then fixed with 3.7% formaldehyde for 10 min. In situ PLA was performed according to the manufacturer’s instructions (Olink Bioscience, Uppsala, Sweden) using the antibodies as described in the text. The interaction of proteins was amplified as distinct bright-red spots and detected using a fluorescence microscope (Personal DV Applied Precision, Issaquah, WA).
Fluorescence microscope
Manufactured by Cytiva
Sourced in United States
A fluorescence microscope is an optical microscope that uses fluorescence to generate an image of the specimen. It illuminates the sample with light of a specific wavelength, causing fluorescent molecules within the sample to emit light of a longer wavelength, which is then detected and used to create an image.
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Lab products found in correlation
3 protocols using fluorescence microscope
1
In Situ Proximity Ligation Assay for Protein Interactions
2
In Situ Protein Interaction Assay
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HCC cells were grown on sterile cover slips, and after 24 h transfection with HA-CPAP/WT or MT [34 (link)] and GFP-HBx, cells were fixed in 3.7% formaldehyde for 10 min and then in situ PLA was performed according to the manufacturer’s instructions (Olink Bioscience, Uppsala, Sweden). Two primary antibodies derived from different species were used to recognize CPAP and GFP. Secondary antibodies were species-specific PLA probes. The interaction of proteins was amplified as distinct bright-red spots and detected using a fluorescence microscope (Personal DV Applied Precision, Issaquah, WA).
3
Mitochondrial Localization of eNOS
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PAEC were transfected with a GFP tagged eNOS expression plasmid (eNOS-GFP) [44 (link)]. After 48 h transfection, cells were labeled with 100 nM MitoTracker (cat#: M22425, Invitrogen, Carlsbad, CA) for 30 min. This dye is not dependent on the mitochondrial membrane potential. The cells were then treated with TGF-β1 (5 ng/ml, 8 h) or GW9662 (5 μM, 24 h) and imaged using a fluorescence microscope (Applied Precision, Issaquah, WA, USA). Mitochondrial localization of eNOS was determined by calculating the Pearson product moment correlation coefficient [45 (link)] between images (green for eNOS-GFP and red for MitoTracker) using FITC (excitation 490nm/emission 528 nm) and TRITC (excitation 555nm/emission 617 nm). Magnification used was 60×.
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