GenePharma was used to design the FISH probes of KCNQ1OT1. T24 and HT-1197 cells were immobilized for 10 min in 4% PFA and washed with PBS (Sigma-Aldrich) for three times. Subsequently, PBS containing with 0.5% Triton X-100 was applied for cell permeabilization. Following washing, pre-hybridization buffer (Sigma-Aldrich) was added into each well to incubate, which lasted for half an hour. The slide was hybridized with KCNQ1OT1 FISH probe, and then was washed three times with 2× Saline Sodium Citrate (SSC; Sigma-Aldrich) at 42 °C. Hoechst 33342 solution (Invitrogen) was used to stain cells. Finally, fluorescent signal was observed by using the microscope [20 (link)].
Saline sodium citrate ssc
Manufactured by Merck Group
Saline-sodium citrate (SSC) is a buffer solution commonly used in various laboratory techniques. It is a mixture of sodium chloride and sodium citrate, which helps maintain a specific pH and ionic strength in the solution. SSC is primarily used as a medium for storing and diluting nucleic acid samples, such as DNA and RNA, to preserve their integrity and stability.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Pre hybridization buffer, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Hybridization solution, by Merck Group (1 mentions)
5 protocols using saline sodium citrate ssc
1
FISH Probing of KCNQ1OT1 in T24 and HT-1197 Cells
2
Visualizing SLC7A11-AS1 and β-TRCP1 Colocalization
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Biotin-labeled SLC7A11-AS1 probe (1,420 nt in exon 3) was generated as described above. Cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 5 min and hybridization with biotin-labeled SLC7A11-AS1 probe in 2× saline sodium citrate (SSC; Sigma) at 65°C overnight in a moist chamber.
For co-localization study, cells were co-stained with rabbit anti-β-TRCP1 antibody (1:100 dilution; ABclonal, China) and streptavidin, Alexa Flour 555 conjugate (1:200 dilution; Thermo Fisher) at 37°C for 1 h, then washed with PBS three times, and incubated with Chicken anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200 dilution, Thermo Fisher) for 2 h at room temperature. The nuclei were counterstained with DAPI (Sigma). The fluorescence images were taken by confocal microscope.
For co-localization study, cells were co-stained with rabbit anti-β-TRCP1 antibody (1:100 dilution; ABclonal, China) and streptavidin, Alexa Flour 555 conjugate (1:200 dilution; Thermo Fisher) at 37°C for 1 h, then washed with PBS three times, and incubated with Chicken anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200 dilution, Thermo Fisher) for 2 h at room temperature. The nuclei were counterstained with DAPI (Sigma). The fluorescence images were taken by confocal microscope.
3
HOXC-AS1 RNA Expression Visualization
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Design and synthesis of HOXC-AS1-FISH probe were accomplished by Invitrogen. BGC-823 or AGS cells were plated on culture slides, fixed in paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA), followed by the sealing with prehybridization buffer (Sigma-Aldrich). Hybridization mixture was added with FISH probe. Slides were washed in buffer adding saline sodium citrate (SSC; Sigma-Aldrich). Cell nuclei were stained by DAPI (Sigma-Aldrich). Cells were examined with Olympus fluorescence microscope (Olympus, Tokyo, Japan).
4
NORAD Expression Analysis in Cell Lines
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HSC-4 and UM1 cells were washed with PBS after being inoculated to glass coverslips in 24-well plates, followed by the fixation of 30 min in 4% formaldehyde (Sigma-Aldrich). After the permeabilization in 70% ethanol (Sigma-Aldrich) overnight, cells were rinsed twice by PBS. Hybridization solution (Sigma-Aldrich) as well as fluorescently labeled NORAD probe (GenePharma) was added during the overnight incubation. Three hours after hybridization, DAPI was adopted to stain cell nuclei and the cells were washed with saline-sodium citrate (SSC; Sigma-Aldrich). In the end, fluorescence microscope was utilized to observe and analyze the stained cells (Olympus Corp., Tokyo, Japan).
5
SKOV3 and OVCAR3 Cells Transcriptional Analysis
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SKOV3 and OVCAR3 cells were added to 24-well plates before being rinsed with PBS, along with fixation by using 4% formaldehyde, followed by permeabilized in 70% ethanol. Later, cells were cleaned with PBS twice and then hybridization solution with fluorescently labeled OIP5-AS1 probe was added for further incubation overnight. Following washing using saline-sodium citrate (SSC; Sigma-Aldrich), cells were dyed in DAPI and finally photographed via a fluorescence microscope (Olympus).
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