Prehybridization buffer

Manufactured by RiboBio

Sourced in China

Prehybridization buffer is a solution used in molecular biology techniques, such as Northern blotting and in situ hybridization, to prepare the target samples for the hybridization step. The buffer helps to block non-specific binding sites and create an optimal environment for the subsequent hybridization process.

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Lab products found in correlation

Confocal fluorescence microscope, by Olympus (1 mentions) Hybridization buffer, by RiboBio (1 mentions) Fish kit, by RiboBio (1 mentions)

2 protocols using prehybridization buffer

Fluorescent In Situ Hybridization of MT1DP

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Cells were washed with cold PBS three times for 10 min, and then fixed in 4% formaldehyde at room temperature for 10 min, followed by permeabilization in 0.5% Triton-100 at 4 °C for 10 min. Thereafter, cells were washed with PBS for three times prior to prehybridization with prehybridization buffer (Ribobio, Guangzhou, China) at 37 °C for 30 min. Afterward, cells were incubated with synthesized digoxygenin-11-dUTP (DIG)-labelled MT1DP FISH probes at 37 °C in the dark overnight in a humid chamber. Cells were then washed with 0.1% Tween-20/4×SSC for three times at 42 °C for 5 min each time, followed by 2×SSC and 1×SSC washing for 5 min for each at 42 °C. Then, cells were incubated with a FITC-anti-digoxin Ab (Jackson, PA, USA) for 1 h, followed by three washes with PBS, and were finally stained with 4ʹ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 10 min at room temperature. Nuclei were counter-stained DAPI. Immunofluorescence was imaged on a confocal fluorescence microscope (Olympus, Japan).

Gao M., Chen M., Li C., Xu M., Liu Y., Cong M., Sang N, & Liu S. (2018). Long non-coding RNA MT1DP shunts the cellular defense to cytotoxicity through crosstalk with MT1H and RhoC in cadmium stress. Cell Discovery, 4, 5.

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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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