FISH was performed to detect the location of chi-miR-483 using procedures according to an article published previously [39 (link)]. In short, micrometer sections (4 μm thick) were deparaffinized, digested with proteinase K, and hybridized with chi-miR-483 probes labeled with cy3 (red); the negative control was established by replacing the probe with Hybridization Buffer (Servicebio, Wuhan, China). Images were then taken using a positive fluorescence microscope (Nikon, Tokyo, Japan).
Hybridization buffer
Manufactured by Wuhan Servicebio Technology
Hybridization buffer is a solution used in molecular biology techniques, such as northern blotting and in situ hybridization, to facilitate the binding of nucleic acid probes to their target sequences. The buffer is designed to provide the optimal conditions for the hybridization process, which is a critical step in these techniques.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (2 mentions)
Proteinase k, by Wuhan Servicebio Technology (2 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Zkx53, by Olympus (1 mentions)
Cy3 conjugated anti digoxin, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated anti biotin antibodies, by Jackson ImmunoResearch (1 mentions)
Neutral balsam, by Sinopharm (1 mentions)
Nuclear fast red, by Wuhan Servicebio Technology (1 mentions)
5 protocols using hybridization buffer
1
In Situ Localization of chi-miR-483
2
Detecting lncRNA TSPEAR-AS2 in Colorectal Cancer
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To verify the expression of lncRNA TSPEAR-AS2 in CRC, digoxigenin (DIG)-labeled CISH was conducted on tumor tissues. The samples were fixed with 4% paraformaldehyde for 2-12 h. And we prepared paraffin sections for hybridizations. Then, we put the sections in boiling water for 15 min and cooled them at room temperature. The specimens were incubated with Proteinase K (Servicebio) at 37°C for 30 min and rinsed three times in PBS (Servicebio). We conducted prehybridization at 37°C for 1 h in hybridization buffer (Servicebio). Then, the samples were incubated overnight at 37°C with fresh hybridization buffers containing 8ng/ml probes instead of prehybridized buffers. The washed specimens were incubated at room temperature in blocking serum containing BSA for 30 min, followed by incubation at 37°C for 40 min with anti-DIG/AP antibody (Jackson). Probes labeled with DIG resulted in permanent diaminobenzidine (DAB) brown-colored, distinct, dot-shaped signals.
3
Visualizing PVT1 Localization in HCC Cells
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FISH is a technique that can be used to detect and locate specific DNA/RNA sequences in cells. The location of PVT1 in MHCC97H and MHCC97L cells was observed using the FISH assay. Probes labeled with Cy5 for PVT1 were synthesized by Servicebio (Wuhan, China). HCC cells were grown on coverslips and treated with a FISH probe in hybridization buffer (Servicebio, Wuhan, China) for 16 h at 37 °C. The cell nuclei were then stained with DAPI (4′6-diamidino-2-phenylindole). The Olympus BX51 fluorescence microscope (Tokyo, Japan) was used to capture these images. Supplementary Table 3 contained the displayed probe sequences.
4
Circular RNA Localization in Cells
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In brief, cells exposed to different treatments were plated in 48‐well plates at a density of 2.5 × 104 cells/well. Next day, the cells were hybridized in hybridization buffer (Servicebio Technology, Wuhan, China) with digoxin (Dig)‐ and biotin (Bio)‐labelled single‐stranded DNA probes at 37°C overnight. Then, the digoxin‐labelled probes (5′‐DIG‐ATAGT TATAA AAAGC TTCAC CCGGA CGG‐DIG‐3′) specific to has‐circ‐0020394 back‐splice region were added (Servicebio Technology), following with Cy3‐conjugated anti‐digoxin and FITC‐conjugated anti‐biotin antibodies (Jackson ImmunoResearch Inc, West Grove, PA). In addition, the sell nuclei were stained with Hoechst. At last, the results were obtained on a fluorescence microscope (ZKX53; Olympus).
5
Evaluating GIHCG Expression in SYSU HCC Samples
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To evaluate the expression level of GIHCG, CISH was applied to SYSU HCC samples. All slices were fixed for 2–12 hrs with 4% paraformaldehyde (DEPC, Servicebio, Wuhan, China), incubated in proteinase K (Servicebio) for 30 mins and rinsed three times with PBS (Servicebio). Next, the samples were prehybridized in hybridization buffer (Servicebio) at 37°C for 1 hr. Then, fresh hybridization buffer containing 8 ng/mL of the corresponding probe was used to replace the prehybridization buffer, and the samples were incubated at 37°C overnight. Finally, nuclear fast red (Servicebio) staining was performed to visualize the cell nuclei of the samples, and the samples were mounted in neutral balsam (Sinopharm Chemical Reagent Co., Ltd) and examined by bright-field microscopy. Based on the staining intensity, the patients were divided into high- or low-expression groups as previously described.22 (link)
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