The subcellular localization of lncRNA DLX6-AS1 was predicted using a bioinformatics tool (http://lncatlas.crg.eu/ ). FISH was carried out to confirm the subcellular localization of lncRNA DLX6-AS1 in LCSCs. According to the instructions provided by the Ribo™ lncRNA FISH probeMix (RiboBio company, Guangzhou, China), Huh7 cells were cultured in cover glasses and transferred to 6-well plates, allowing them to incubate for one day in order let the cell confluence reach approximately 80%. Next, cells were fixed in 1 mL 4% paraformaldehyde at room temperature, followed by treatment with 2 μg/mL protease K, glycine, and ethyl phthalate reagent. After that, cells were incubated with 250 μL prehybridization solution at 42 °C for 1 h, and then incubated with 250 μL of 300 ng/mL hybridization solution containing probe at 42 °C overnight after removing the prehybridization solution. The cells were then washed with phosphate-buffered saline with Tween-20 (PBST), incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1: 800) for five minutes in a 24-well plate, followed by a PBST rinse. Finally, cells were sealed with anti-fluorescence quenching agent. By randomly selecting give different visual fields, cells were observed and photographed under a fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan).
Ribo lncrna fish probe mix
Manufactured by RiboBio
Sourced in China
Ribo™ lncRNA FISH Probe Mix is a laboratory reagent designed for the detection and visualization of long non-coding RNA (lncRNA) molecules using fluorescence in situ hybridization (FISH) techniques. The product provides a set of probes that target specific lncRNA sequences, enabling researchers to study the localization and expression patterns of these important regulatory RNA molecules.
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Lab products found in correlation
Ribo fluorescent in situ hybridization kit, by RiboBio (4 mentions)
Paris kit, by Thermo Fisher Scientific (4 mentions)
Fluorescence microscope, by Olympus (4 mentions)
Fluorescent in situ hybridization kit, by RiboBio (2 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Ribo fish kit, by RiboBio (1 mentions)
Cr0013, by Sparkjade (1 mentions)
S1030, by Solarbio (1 mentions)
D9542, by Merck Group (1 mentions)
18s fish probes, by RiboBio (1 mentions)
Ma0192, by Meilun (1 mentions)
13 protocols using ribo lncrna fish probe mix
1
Subcellular Localization of lncRNA DLX6-AS1 in LCSCs
2
Visualizing lnc-EST12 Expression in RAW264.7 Cells
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The red fluorescent Cy3-labeled probe (Ribo-lncRNA FISH Probe Mix) against lnc-EST12 was designed by RiboBio Company (Guangzhou, China) and was detected using a Fluorescent In Situ Hybridization Kit (RiboBio, China) according to the manufacturer’s instructions. Briefly, RAW264.7 cells grown on cover slips in 24-well plates with the indicated treatment were fixed with 4% (v/v) paraformaldehyde for 10 min at room temperature and then washed three times with cold PBS. The cells were permeabilized in PBS containing 0.5% Triton X-100 for 5 min at 4 °C and then blocked with prehybridization buffer for 30 min at 37 °C. Cells were then incubated with a probe in hybridization buffer (2.5 μL, 20 μM probe in 250 μL hybridization buffer) overnight at 37 °C in the dark. After hybridization, the cells were washed in the dark with washing buffer (4 × SSC/2 × SSC/1 × SSC) and then stained with DAPI for 10 min. The cells were washed three times with PBS and then imaged with a ZEISS confocal microscope under an oil objective.
3
Long Non-coding RNA FISH Protocol
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Ribo™ lncRNA FISH Probe Mix (red, C10920, Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China) was employed in the current study. Cells (6 × 104 cells/well) were seeded in a 24-well plate and fixed with 1 mL of 4% paraformaldehyde for 10 min. Pre-cooled PBS containing 0.5% Triton X-100 (1 mL) was added to each well and allowed to stand at 4 °C for 5 min. Next, the pre-hybridization solution (200 μL) was added and sealed at 37 °C for 30 min. After removal of the pre-hybridization solution, the hybridization solution containing anti-SNHG14 oligonucleotide probes (Wuhan GeneCreate Biological Engineering Co., Ltd., Wuhan, Hubei, China) was added to each well and allowed to hybridize overnight at 37 °C in dark conditions. On the following day, the cells were washed with cleansing solution I at 42 °C (4 × saline sodium citrate (SSC), 0.1% Tween-20), cleansing solution II (2 × SSC), cleansing solution III (1 × SSC) and 1 × PBS successively. Cells were then stained with 4′,6-diamidino-2-phenylindole at a ratio of 1: 800 for 10 min and mounted with nail polish. Finally, the cells were observed and imaged in 5 random fields under a fluorescence microscope (Olympus, Tokyo, Japan).
4
Subcellular Localization of lncRNA by FISH
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The subcellular localization of lncRNA was evaluated by FISH assay using a Ribo Fluorescent In Situ Hybridization Kit (Ribobio, Guangzhou, China). Briefly, HCC cells were planted into 24-well plate at 8 × 104 (HepG2) or 4 × 104 (QGY-7703) per well. The transfected HCC cells were fixed in 4% paraformaldehyde and incubated in 0.5% Triton X-100 for higher membrane permeability. After prehybridization, the cells were incubated with Ribo lncRNA FISH Probe Mix (Ribobio) at 37 °C overnight. After stringency washing and DAPI staining, fluorescence was observed under an IX71 fluorescence microscope (Olympus, Shinjuku, Japan).
5
Detecting HOTAIR Subcellular Localization
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FISH was conducted to detect the subcellular localization of HOTAIR using the RibolncRNA FISH Probe Mix (C10920, Red, Guangzhou RiboBio, Guangzhou, Guangdong China), according to the manufacturer’s instructions. Specifically, the cells were plated onto coverslips placed in 24-well plates, at a density of 6 × 104 cells/well and cultured until they reached a 60%–70% confluence. After fixation in 4% paraformaldehyde (1 mL) at room temperature for 10 min, each well was blocked with 1 mL pre-cooled permeation solution containing 0.5% Triton X-100 PBS at 4°C for 5 min. Then the cells were incubated with 200 uL prehybridization solution at 37°C for 30 min and hybridized with hybridization solution containing the probe (Wuhan Jin Kairui Bioengineering, Wuhan, China) at 37°C overnight in an environment devoid of light. The cells were then stained with 4’, 6-diamidino-2-phenylindole (DAPI, 1:800) for 10 min and sealed with nail polish. Finally, 5 visual fields were selected and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
6
Cytoplasmic and Nuclear RNA Isolation
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LoVo and SW480 cells were fixed in 4% formaldehyde for 10 minutes and washed with phosphate-buffered saline (PBS) for 5 minutes. Fixed cells were permeabilized for 10 minutes in PBS containing 0.5% Triton-X 100, followed by incubation at 37°C with pre-hybridization solution for 30 minutes. The cells were then incubated overnight at 37°C with probe-hybridization solution, and each slide was washed with hybridization washing buffer and dehydrated. The air-dried slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) for detection. Ribo FISH kit and Ribo lncRNA FISH probe mix were purchased from Ribo (Guangzhou, China). The sequences of the probes used in this study are presented in Table 1 . Cytoplasmic and nuclear RNA were isolated using a PARIS kit (Life Technologies) according to the manufacturer’s protocol.
7
Subcellular Localization of lncRNA
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FISH assay was performed using a Ribo™ Fluorescent In Situ Hybridization Kit and Ribo™ lncRNA FISH Probe Mix (Ribo, Guangzhou, China) according to the manufacturer’s protocols. The separation of nuclear and cytosolic fractions was performed using a PARIS Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
8
Ribo FISH Assay for Subcellular Localization
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The FISH assay was conducted with Ribo Fluorescent In Situ Hybridization Kit and Ribo lncRNA Fish Probe Mix (Ribo, Guangzhou, China). The separation of nucleus and cytoplasm was conducted using PARIS Kit (Life Technologies, Carlsbad, USA) in line with specifications.
9
LINC00882 Expression Profiling in HCC
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Expressions of LINC00882 in tissue microarrays containing HCC specimen and noncancerous liver specimen were detected by in situ hybridization. Three probes targeting different LINC00882 regions (Ribo™ lncRNA FISH Probe Mix; RiboBio, Guangzhou, People’s Republic of China) were synthesized and labeled with DIG-dUTP at the 3′ end. GAPDH probes (lnc110102 h-18S FISH Probe Mix; RIBO Biotech, Guangzhou, People’s Republic of China) were used as controls. A scoring criterion, which counts both the staining intensity and the number of positively stained cells, was used for semi-quantification of in situ hybridization. The scoring was graded as 0 (negative), 1 (<10% positive), 2 (10%–50% positive) or 3 (>50% positive) according to the positively stained proportion and staining intensity of cells. The final scores ranged from 0 to 1, which were regarded as low expression, and scores 2–3 were regarded as high expression. Five representative visual fields were selected randomly by two pathologists to score.
10
Detecting lncRNA Expression via FISH
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The red fluorescence-labeled probe (Ribo-lncRNA FISH Probe Mix) against EDAL lncRNA or 18S was designed by Ribobio Co (Guangzhou, China) and was detected by Fluorescent In Situ Hybridization Kit (Ribobio, R11060.1) according to the manufacturer’s instructions. Briefly, N2a cells grown on cover slips in 24-well plates with indicated treatment were fixed with 4% (v/v) paraformaldehyde for 10 min (min) at room temperature then washed three times with cold PBS. And the cells were permeabilized in PBS containing 0.5% Triton X-100 for 5 min in 4 °C, then blocked in pre-hybridization buffer for 30 min at 37 °C. Cells were then incubated with a hybridization buffer-containing probe (2.5 μl, 20 μM probe in 250 μl hybridization buffer) overnight at 37 °C away from the light. After hybridization, cells were washed in the dark with washing buffer (4 × SSC/2 × SSC/1 × SSC) then stained with DAPI for 10 min. Cells were again washed three times with PBS, and then imaged with a ZEISS confocal microscope under oil objective.
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