FISH assays were performed using RiboTM Fluorescence In Situ Hybridization Kit (RiboBio) under the manufacturer’s instructions. Cy3-labeled probes targeting circFAM192A, U6, 18S were purchased from RiboBio.
Ribo fluorescence in situ hybridization kit
Manufactured by RiboBio
Sourced in China
The Ribo fluorescence in situ hybridization (FISH) kit is a laboratory tool used for the detection and localization of specific ribonucleic acid (RNA) sequences within cells or tissue samples. The kit provides reagents and protocols for performing FISH, a molecular biology technique that employs fluorescently labeled probes to visualize and analyze the distribution and expression of target RNA molecules.
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Lab products found in correlation
Confocal laser scanning microscope, by Leica (1 mentions)
Lsm5 confocal microscope, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Confocal microscope, by Philips (1 mentions)
Cytoplasmic nuclear rna purification kit, by Norgen Biotek (1 mentions)
Tcs sp8, by Leica (1 mentions)
7 protocols using ribo fluorescence in situ hybridization kit
1
Fluorescent In Situ Hybridization Assay
2
Detecting Podocyte mRNA Expression Using RNA-FISH
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RNA-FISH was performed with a RiboTM Fluorescence In Situ Hybridization Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. In brief, mature podocytes were cultured at appropriate densities on slides on the bottoms of the wells of 24-well plates. Then, 4% paraformaldehyde was added and incubated for 5 min to fix the different podocytes when the cell confluence reached 60%–70%, and then the cells were incubated with 0.5% Triton X-100 for 5 min. Then, 200 μL prehybridization solution was added to the cells and incubated at 37°C for 30 min in the dark. The probe hybridization solution was prepared by adding the Miat FISH Probe or Internal Reference FISH Probe to the hybridization solution, which was preheated at 37°C. The prehybridization solution was discarded, and 100 μL probe hybridization solution was added and incubated overnight at 37°C to hybridize the podocytes. The cells were carefully washed 3 times with hybridization wash solutions I, II, and III at 42°C in the dark. Images were observed and captured by confocal laser scanning microscope (Leica, Wetzlar, Germany) after the nuclei were stained for 10 min with a 4,6-diamidino-2-phenylindole (DAPI) working solution.
3
FISH Assay for Circular RNA Detection
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FISH assays were conducted with RiboTM Fluorescence In Situ Hybridization Kit (RiboBio) under the manufacturer’s instruction. Cy3-labeled probes targeting hsa_circ_0007967, U6, 18S were purchased from RiboBio. Cells were seeded into eight-well plate and incubated for 12 h before fixation. After 30 min’ fixation, and 10 min’ permeabilization (0.5% Triton X-100), cells were prehybridized in prehybridization buffer at 37 °C for half an hour. Then cells were hybridized in hybridization buffer with specific probes at 37 °C overnight in the dark. 4×SSC (including 0.1% Tween-20), 2×SSC and 1×SSC were used for washing off hybridization buffer at 42 °C in the dark. Confocal images were captured by Zeiss LSM5 confocal microscope (Carl Zeiss Jena, Oberkochen, Germany).
4
RNA FISH Protocol for Gene Expression
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FISH analysis was performed using Ribo ™ fluorescence in situ hybridization kit (Ribobio Company, China). The counting probe was purchased from Empire Genomics and labeled with fluorescent dye. RNA FISH was performed using a fluorescence in situ hybridization kit in accordance with the manufacturer’s instructions. Fluorescence detection was carried out by AxioObserver Z1 microscope (Zeiss).
5
RNA-FISH and Fractionation Assay
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RNA-FISH was conducted using a Ribo fluorescence in situ hybridization kit (C10910, RiboBio, China) in accordance with the manufacturer’s directions. circCCDC134 and hsa-miR-503-5p FISH probes were designed and synthesized by RiboBio. In brief, the cells were seeded, fixed with 4% paraformaldehyde and incubated with a hybridized 5 mM probe at 37 °C overnight. All images were visualized and obtained under a confocal microscope (Philips).
In total, 1 × 106 cells were used for the RNA fractionation assays. RNA from the nucleus and cytoplasm was separated by a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp, Canada) following the manufacturer’s instructions.
In total, 1 × 106 cells were used for the RNA fractionation assays. RNA from the nucleus and cytoplasm was separated by a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp, Canada) following the manufacturer’s instructions.
6
FISH-based Linc00707 Expression Analysis
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The expression and localization of Linc00707 in TNBC cells were detected by FISH method. Briefly, cells were grown on cover slides, fixed in 4% formaldehyde for 10 min at room temperature, then cleaned with PBS for 3 times, use 0.5% Triton permeable membrane, then cleaned with PBS for 3 times, Ribo fluorescence in situ hybridization kit (RiboBio, Guangzhou, China) was used for FISH detection. Cy3-labeled probes are resistant to Linc00707 or control probes are resistant to U6 snRNA and 18 S rRNA. Representative images were collected and analyzed under a confocal laser scanning microscope (TCS SP8, Leica, Germany). All experiments were independently repeated three times.
7
RNA FISH Assay Using RiboBio Kit
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The Ribo™ Fluorescence In Situ Hybridization Kit (RiboBio, Guangzhou, China) was used to perform RNA fluorescence in situ hybridization. All the manipulations were performed according to the manufacturer’s protocols. Probes that specifically target ZXF1 were designed and synthesized by RiboBio.
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