The co-recruitment assay was done as previously described (48 (link)). In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells grown on coverslips were fixed in PBS 1X/paraformaldehyde 4% and permeabilized with a PBS 1X/ Triton X100 0.1% solution. Primary antibodies were diluted in 1x antibody diluent and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed twice for 5 min in PBS 1X. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain for nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged. Images were processed and assembled with Photoshop CS5 (Adobe). The antibodies used in these experiments were as follows: anti-SMN (2B1), anti-Gemin2 (2E17), anti-Gemin3 (44 ), anti-Gemin4 (45 (link)), anti-Gemin6 (12307–2-AP, PTG Lab), anti-Gemin7 (6E2, Millipore), anti-Gemin8 (1F8) (20 (link)), anti-NUFIP (12515–1-AP, Proteintech) and anti-GAPDH (Abcam).
Sp5 aobs x confocal microscope
Manufactured by Leica
The Leica SP5-AOBS X is a confocal microscope designed for high-performance imaging. It features a fully automated, tunable Acousto-Optical Beam Splitter (AOBS) that allows for flexible control of excitation and emission wavelengths. The microscope is equipped with multiple laser lines and photomultiplier tube (PMT) detectors to enable simultaneous acquisition of multiple fluorescent signals.
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Lab products found in correlation
3 protocols using sp5 aobs x confocal microscope
1
Proximity Ligation Assay for SMN Complex
2
In Situ Proximity Ligation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells grown on coverslips were fixed in 1× PBS, 3% paraformaldehyde during 20 min and permeabilized for 5 min in a 1× PBS, 0.1% Triton X-100 solution. Primary antibodies were diluted in 1× antibody dilution buffer and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed three times for 5 min in 1× PBS. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. Finally, cells were incubated for 20 min with Alexa Fluor™ 546 Phalloidin (Thermo Fischer scientific) to detect the cytoskeleton. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged with the ImageJ software.
+ Expand
In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells grown on coverslips were fixed in 1× PBS, 3% paraformaldehyde during 20 min and permeabilized for 5 min in a 1× PBS, 0.1% Triton X-100 solution. Primary antibodies were diluted in 1× antibody dilution buffer and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed three times for 5 min in 1× PBS. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. Finally, cells were incubated for 20 min with Alexa Fluor™ 546 Phalloidin (Thermo Fischer scientific) to detect the cytoskeleton. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged with the ImageJ software.
3
In situ Proximity Ligation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells grown on coverslips were fixed in 1x PBS, 3% paraformaldehyde during 20 min and permeabilized for 5 min in a 1x PBS, 0.1% Triton X-100 solution.
Primary antibodies were diluted in 1x antibody dilution buffer and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed three times for 5 min in 1x PBS. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. Finally, cells were incubated for 20 min with Alexa Fluor™ 546 Phalloidin (Thermo Fischer scientific) to detect the cytoskeleton. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged with the ImageJ software.
Primary antibodies were diluted in 1x antibody dilution buffer and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed three times for 5 min in 1x PBS. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. Finally, cells were incubated for 20 min with Alexa Fluor™ 546 Phalloidin (Thermo Fischer scientific) to detect the cytoskeleton. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged with the ImageJ software.
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