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Hybridization buffer

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Hybridization buffer is a solution used in molecular biology techniques, such as Northern blotting and in situ hybridization, to facilitate the binding of labeled nucleic acid probes to their complementary target sequences. It provides the necessary conditions for efficient hybridization between the probe and the target.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions) Fish kit, by RiboBio (2 mentions) Fv10 asw viewer, by Olympus (1 mentions) Fv1000, by Olympus (1 mentions) Fish probe, by RiboBio (1 mentions) Prehybridization buffer, by RiboBio (1 mentions) Tcs sp8 confocal microscope, by Leica (1 mentions) Confocal laser scanning microscopy, by Zeiss (1 mentions) Ab33589, by Abcam (1 mentions) Ab104139, by Abcam (1 mentions) Rna probes, by RiboBio (1 mentions) Alexa fluor 594, by Abcam (1 mentions)

7 protocols using hybridization buffer

1

Visualizing lncRNA VIM-AS1 Expression

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FISH assays were performed using a RiboTM lncRNA FISH probe Mix Kit (cat. no. c10910; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Oligonucleotide modified Cy-3-labeled probes for VIM-AS1 (5′-TAG GAC TTC CTA GTA CTT CTG A-3), GAPDH and U6 were designed and synthesized by Genecreate. C4-2 cells were seeded on 20-mm confocal dishes (Corning, Inc). After overnight incubation, C4-2 cells were fixed with 4% paraformaldehyde for 20 min at 4°C and permeabilized using Triton X-100 for 90 sec at 4°C. Next, 250 µl prehybridization solution with 1% blocking solution (Guangzhou RiboBio Co., Ltd.) was added to C4-2 cells and cells were incubated at 42°C for 1 h. Subsequently, C4-2 cells were incubated with 100 µl hybridization buffer (Guangzhou RiboBio Co., Ltd.) supplemented with 1% blocking solution and 2.5 µl 20 µM 22-nucleotide CY-3-labeled-VIM-AS1, CY-3-labeled-GAPDH or CY-3-labeled-U6 FISH probe at 37°C overnight in a dark moist chamber. The following day, cells were washed three times in 2X sodium citrate buffer for 5 min at 42°C and stained with DAPI at 4°C for 10 min. Images were acquired using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at a magnification of ×400.
Shi S.J., Han D.H., Zhang J.L., Li Y., Yang A.G, & Zhang R. (2023). VIM-AS1 promotes proliferation and drives enzalutamide resistance in prostate cancer via IGF2BP2-mediated HMGCS1 mRNA stabilization. International Journal of Oncology, 62(3), 34.
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2

GAS5 Expression Visualization in HASMCs

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HASMCs on coverslips were fixed in 4% paraformaldehyde, washed 3 times with PBS and then permeabilized with 0.2% Triton X-100 in PBS for 30 minutes. The HASMCs were hybridized with a hybridization buffer (RiboBio, Guangzhou, China) and incubated with a labeled GAS5 probe overnight at 37°C. The GAS5 probe was purchased from BersinBio (Guangzhou, China). The cells were then washed with 2× SSC, 1× SSC, and 0.5× SSC and incubated with a mouse anti-digoxin antibody conjugated with AP (Boster Biotechnology Co., Ltd., Wuhan, China). Subsequently, the cells were incubated in DAPI (Beyotime Biotechnology, Shanghai). Images were obtained using aLeica (TCS Sp8) confocal microscope.
He X., Wang S., Li M., Zhong L., Zheng H., Sun Y., Lai Y., Chen X., Wei G., Si X., Han Y., Huang S., Li X., Liao W., Liao Y, & Bin J. (2019). Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis. Theranostics, 9(19), 5558-5576.
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3

CARMN Expression Detection Protocol

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Hybridization was carried out on the sections using hybridization buffer (RiboBio, China) and incubated them with a labeled CARMN probe overnight. Post-hybridization, the sections were purified using SSC and subsequently incubated with both primary antibody and secondary antibodies. The final step involved incubation in DAPI. The sequences of the human and mouse CARMN probes are detailed in Supplemental Table 4.
Liu S., Zhou H., Han D., Song H., Li Y., He S., Du Y., Wang K., Huang X., Li X, & Huang Z. (2024). LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF. Cellular and Molecular Life Sciences: CMLS, 81(1), 175.
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4

FISH Assay for Detecting Cellular Markers

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This assay was performed with a FISH kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Briefly, fixed cells were further incubated with 20 μM FISH probe in hybridization buffer (RiboBio) at 37°C overnight. Then, the cells were stained with DAPI, and fluorescence signals were detected using a fluorescence microscope.
Wu S., Cui Y., Zhao H., Xiao X., Gong L., Xu H., Zhou Q., Ma D, & Li X. (2022). Trophoblast Exosomal UCA1 Induces Endothelial Injury through the PFN1-RhoA/ROCK Pathway in Preeclampsia: A Human-Specific Adaptive Pathogenic Mechanism. Oxidative Medicine and Cellular Longevity, 2022, 2198923.
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5

FISH Assay for NMCM Visualization

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Fluorescence in Situ Hybridization (FISH) Kit (Ribo, China) was performed, as in manufacturer’s instructions. In brief, the NMCM, grown on slides, were fixed with 4% formaldehyde for 10 minutes at room temperature, followed by three washes with PBS (phosphate buffer saline) and 5 minutes of permeabilization with 0.5% Triton X-100. Then, the cells were washed with PBS thrice. Before hybridization, the cells were blocked with pre-hybridization buffer (Ribo), containing the blocking solution (99:1) at 37°C for 30 minutes. Then, 0.5 mM Ahit FISH probe was hybridized in hybridization buffer (Ribo) at 37°C overnight. After hybridization, the cells were washed with 4X sodium citrate buffer (SSC) for 5 minutes thrice and 2X SSC, 1X SSC once at 42°C. The nuclei were stained with DAPI while NMCM microfilaments were stained with α-actinin. After washing with PBS and sealing, photographs were taken by confocal microscopy. Ahit FISH probe was GGACATGTGTCCACAGTGTCCATACACCTTGCTC.
Yu J., Yang Y., Xu Z., Lan C., Chen C., Li C., Chen Z., Yu C., Xia X., Liao Q., Jose P.A., Zeng C, & Wu G. (2020). Long Noncoding RNA Ahit Protects Against Cardiac Hypertrophy through SUZ12-Mediated Downregulation of MEF2A. Circulation. Heart failure, 13(1), e006525.
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6

Visualizing circChrodc1 in Human Aortic VSMCs

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Human aortic VSMCs on coverslips were fixed with 4% paraformaldehyde, washed 3 times with PBS, and then permeabilized with 0.2% Triton X-100 in PBS for 30 min. Human aortic VSMCs were hybridized with hybridization buffer (RiboBio, Guangzhou, China) and incubated with a labeled human circChrodc1 probe (5′-TATGTGCAAGCATCTTGGCCT-3′) overnight at 37°C. The circChrodc1 probe was purchased from BersinBio (Guangzhou, China). The cells were then sequentially washed with 2× SSC, 1× SSC, and 0.5× SSC and incubated with a mouse anti-digoxin antibody conjugated with AP (Boster Biotechnology, Wuhan, China). Subsequently, the cells were incubated with DAPI (Beyotime Biotechnology, Shanghai). Images were obtained using a Leica (TCS Sp8) confocal microscope.
He X., Li X., Han Y., Chen G., Xu T., Cai D., Sun Y., Wang S., Lai Y., Teng Z., Huang S., Liao W., Liao Y., Bin J, & Xiu J. (2021). CircRNA Chordc1 protects mice from abdominal aortic aneurysm by contributing to the phenotype and growth of vascular smooth muscle cells. Molecular Therapy. Nucleic Acids, 27, 81-98.
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7

RNA-FISH Detection of circHipk3 and miR-133a

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Cy3-labeled RNA probes targeting the circHipk3 backsplice sequence and miR-133a were purchased from RiboBio (Guangzhou, China) and used in RNA-FISH assays, performed as described previously.44 (link) Briefly, cultured cells were washed with PBS, permeabilized with 0.5% Triton X-100, and incubated with RNA probes in hybridization buffer (RiboBio, Guangzhou, China). Then, cells were incubated with anti-cTnT (ab33589; Abcam), followed by fluorescent secondary antibodies (Alexa Fluor 594; Abcam) and DAPI staining (ab104139; Abcam). Images were captured using confocal laser-scanning microscopy (Carl Zeiss).
Si X., Zheng H., Wei G., Li M., Li W., Wang H., Guo H., Sun J., Li C., Zhong S., Liao W., Liao Y., Huang S, & Bin J. (2020). circRNA Hipk3 Induces Cardiac Regeneration after Myocardial Infarction in Mice by Binding to Notch1 and miR-133a. Molecular Therapy. Nucleic Acids, 21, 636-655.
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