The Duolink II fluorescence assay was used to analyze the interaction between YY1AP1 and YY1 in the HepG2-Tet-C2 cells. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 10 minutes, then blocked and incubated with mouse anti-YY1AP1 Ab (1:100 dilution; Sigma) and rabbit anti-YY1 Ab (1:100 dilution; Abcam) overnight at 4°C. The Duolink II Proximity Ligation Assay (PLA) was then performed according to manufacturer's manual (Olink Bioscience, Uppsala, Sweden). A reporter substrate is formed if the PLA® probes are in very close proximity to one another indicating an interaction between the proteins examined. The reporter substrate appears as a red dot under a standard microscope. After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss). Ten images per condition were acquired at 100× magnification with an average of ten cells per image. The number of red dots per cell was quantified from the average number of red dots per cell in ten images.
Multiphoton confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Multiphoton confocal laser-scanning microscope is a specialized imaging system that uses a focused laser beam to excite multiple photons within a sample, enabling high-resolution, three-dimensional imaging of biological specimens. The core function of this microscope is to provide a non-invasive, high-sensitivity method for visualizing the internal structure and dynamics of living cells and tissues.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Mouse anti yy1ap1 ab, by Merck Group (2 mentions)
Rabbit anti yy1 ab, by Abcam (2 mentions)
Duolink 2 proximity ligation assay, by Olink (2 mentions)
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dapi blue, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Alexa fluor 488 or 594 dye conjugate, by Thermo Fisher Scientific (1 mentions)
7 protocols using multiphoton confocal laser scanning microscope
1
Investigating Protein-Protein Interactions with Duolink II
2
Transient Expression of BdCBF1-GFP in Brachypodium Protoplasts
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The GFP-coding sequence was fused in-frame to the 3′ end of BdCBF1 cDNA sequence, and the gene fusion was expressed transiently in Brachypodium protoplasts, as described previously
[34 (link)]. After incubation for 16 h at room temperature in complete darkness, the protoplasts were observed using the Multi-photon Confocal Laser Scanning microscope (Carl Zeiss, Jena, Germany).
[34 (link)]. After incubation for 16 h at room temperature in complete darkness, the protoplasts were observed using the Multi-photon Confocal Laser Scanning microscope (Carl Zeiss, Jena, Germany).
3
Detecting CXCR4 Expression in Trastuzumab-Resistant Cells
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For comparing CXCR4 expression in acquired trastuzumab-resistant cells or their parental cells, the cells were grown on coverslips pre-coated with polylysine and fixed in 4% paraformaldehyde for immunofluorescent staining. For testing the effect of AMD3100 on CXCR4 translocation induced by SDF-1α, the trastuzumab-resistant cells were treated with AMD3100 for 48 hours. After serum starvation overnight, the cells received SDF-1α stimulation for the designed time. For immunofluorescent staining, cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes and permeabilized in 0.25% Triton X-100 for 5 minutes. After blocking with 3% bovine serum albumin for 1 hour, cells were incubated with the CXCR4 antibody overnight at 4°C, followed by staining with Alexa Fluor 488–conjugated goat anti-mouse secondary antibody (Invitrogen). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; Thermo Scientific). After mounting, microscopic images were captured by a multiphoton confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY).
4
Imaging CXCR4 Translocation in Trastuzumab-Resistant Cells
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For comparing CXCR4 expression in acquired trastuzumab-resistant cells or their parental cells, the cells were grown on coverslips pre-coated with polylysine and fixed in 4% paraformaldehyde for immunofluorescent staining. For testing the effect of AMD3100 on CXCR4 translocation induced by SDF-1α, the trastuzumab-resistant cells were treated with AMD3100 for 48 h. After serum starvation overnight, the cells received SDF-1α stimulation for the designed time. For immunofluorescent staining, cells were fixed in 4% paraformaldehyde at room temperature for 20 min and permeabilized in 0.25% Triton X-100 for 5 min. After blocking with 3% bovine serum albumin for 1 h, cells were incubated with the CXCR4 antibody overnight at 4 °C, followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Scientific). After mounting, microscopic images were captured by a multiphoton confocal laser scanning microscope (Carl Zeiss, Thornwood, NY).
5
Immunoprecipitation and Immunocytochemistry Protocols
6
Immunocytochemistry Staining Protocol
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For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized in 5% Triton X-100 for 5 min and then stained using primary antibodies. The secondary antibodies used were anti-mouse Alexa Fluor 488 or 594 dye conjugate and/or anti-rabbit Alexa Fluor 488 or 594 dye conjugate (Life Technologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue; Life Technologies). After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA).
7
Investigating Protein-Protein Interactions with Duolink II
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The Duolink II fluorescence assay was used to analyze the interaction between YY1AP1 and YY1 in the HepG2-Tet-C2 cells. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 10 minutes, then blocked and incubated with mouse anti-YY1AP1 Ab (1:100 dilution; Sigma) and rabbit anti-YY1 Ab (1:100 dilution; Abcam) overnight at 4°C. The Duolink II Proximity Ligation Assay (PLA) was then performed according to manufacturer's manual (Olink Bioscience, Uppsala, Sweden). A reporter substrate is formed if the PLA® probes are in very close proximity to one another indicating an interaction between the proteins examined. The reporter substrate appears as a red dot under a standard microscope. After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss). Ten images per condition were acquired at 100× magnification with an average of ten cells per image. The number of red dots per cell was quantified from the average number of red dots per cell in ten images.
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