The sub-cellular localization of NCK1-AS1 in glioma cell line was assessed according to the protocols of a lncRNA FISH probe Mix (RiboBio). In brief, cover glasses were put into 6-well plates on which glioma cells were seeded. One day later when the cell confluence got to 80%, the cell slides were collected, washed in PBS, and treated with 1 mL 4% paraformaldehyde. Next, the cells were given Protease-K (2 μg/mL), glycine and acetylating agent, and then cultured with prehybridization agent (250 μL) at 42 °C for 1 h, and then with hybridization agent (250 μL) containing probes (300 ng/mL) at 42 °C overnight. After 3 PBST washes, the cells were treated with PBST-DAPI (1:800) for nucleus staining for 5 min. Next, the cell slides were washed in PBST (3 × 3 min), sealed with anti-fluorescence quencher, and then observed under the fluorescence microscope (× 400) with 5 random fields included.
Fish probe mix
Manufactured by RiboBio
Sourced in China
The FISH Probe Mix is a set of fluorescently labeled DNA probes designed for use in Fluorescence In Situ Hybridization (FISH) techniques. The probes are designed to target specific genomic regions or DNA sequences, allowing for the visualization and localization of targeted genetic material within cells or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ribotm fluorescent in situ hybridization kit, by RiboBio (2 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Dmi3000b, by Leica camera (1 mentions)
Triton x 100, by Biosharp (1 mentions)
Axio imager z1 fluorescent microscope, by Zeiss (1 mentions)
Fv1000 laser microscope, by Olympus (1 mentions)
Fish kit, by RiboBio (1 mentions)
5 protocols using fish probe mix
1
Subcellular Localization of lncRNA NCK1-AS1 in Glioma
2
FISH Probe Hybridization in Testes
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FISH probe mix was synthesized by Ribo Bio Technology Co Ltd (Guangzhou, China). FISH was performed with the FISH kit according to the manufacturer's protocol. Testes were fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized in PBS with 1% Triton X‐100 on ice for 30 min. Followed by pretreatment with pre‐hybridization buffer at 37°C for 30 min. Subsequently, testes were hybridized with 20 μM using Cy3‐labeled RNA of FISH probe mix in a moist chamber at 37°C overnight. Testes were rinsed thrice in 4 × SSC with 0.1% Tween‐20 for 5 min at 42°C, washed once for 5 min at 42°C in 2 × SSC and then washed once for 5 min at 42°C in 1 × SSC. After hybridization, testes were stained with Hoechst‐33,342 (Solarbio, 1.0 mg/mL) for 5 min before mounting.
3
RNA-FISH Assay for HOXD-AS2 Localization
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RNA-FISH assay was carried out utilizing RiboTM Fluorescent in Situ Hybridization Kit (RiboBio, China) in accordance with manufacturer’s protocol. Briefly, Bel-7402 cells were paved on the glass coverslips in a 24-well plate. When cells reached 60% confluency, they were fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.5%). The cells were then blocked with prehybridization buffer at 37°C for 40 min and incubated with 20 μM human HOXD-AS2 FISH Probe Mix (Cy3) (lnc1100245; RiboBio, China) at 37°C overnight. After washing by Hybridization Wash Buffer, cell nuclei were counterstained with DAPI. The glass coverslips carried cells were then placed on a glass slide and fluorescent images were observed using a fluorescence microscope (Leica DMI3000B, Germany). U6 and 18S rRNA were adopted as subcellular localization markers to indicate cytoplasmic and nuclear fraction, respectively, and FISH Probe Mix targeting U6 (lnc110101) and 18S (lnc110102) were also acquired from RiboBio (Guangzhou, China).
4
Fluorescent In Situ Hybridization in NSCLC
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NSCLC tumor cells were seeded on glass coverslips, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.5% Triton X-100 (Biosharp, China) for 10 min at room temperature. The slides were then processed using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). The corresponding FISH Probe Mix was also designed by RiboBio Co. The experiment was repeated three times in A549 cells. Images were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany).
5
Spatiotemporal Analysis of MBNL1-AS1 Localization
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IF and FISH assays were performed as previously described [14 (link), 26 (link)]. For FISH, the MBNL1-AS1 subcellular localization was assessed by the lncRNA FISH Probe Mix and FISH kit which was obtained from Guangzhou RiboBio Co., Ltd. The 4% paraformaldehyde-fixed cells were fixed in PBS. After washed by phosphate buffered solution with tween for three times, the anti-fluorescence quencher sealed the indicated cells, and the images were taken by the FV1000 laser microscope (Olympus, Japan). For IF, cells were fixed with 4% formaldehyde and subjected to standard immunofluorescence staining. The anti-CENPA and secondary antibodies were obtained from Thermo Fisher Scientific. The data was analyzed by Image J software. The nuclei were dyed with DAPI. The 3D-cultured spheroids were conducted as previously described [26 (link)].
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