miRNA-126 In Situ Hybridization Protocol

The miRNA-126 ISH was performed essentially as previously described [29 (link),30 (link)]. In brief, 6-μm thick tissue sections were pre-treated with a proteinase-K solution followed by hybridization with a double 6-carboxyfluorescein (FAM)-labelled Locked Nucleic Acid (LNA) miRCURY probe (LNA™ microRNA detection probe, Exiqon A/S, Denmark). After stringent washes in SSC buffers, the probes were detected with alkaline phosphatise-conjugated sheep anti-FAM Fab fragments (Roche). The 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-Indolyl-phosphate (BCIP) substrate (Roche) were added and samples were incubated leading to a blue precipitate (the ISH signal). Counterstaining was performed with nuclear fast red. All slides were processed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland) [30 (link)] in series of 48 slides per run.

Free full text: Click here

Hansen T.F., Nielsen B.S., Jakobsen A, & Sørensen F.B. (2015). Intra-tumoural vessel area estimated by expression of epidermal growth factor-like domain 7 and microRNA-126 in primary tumours and metastases of patients with colorectal cancer: a descriptive study. Journal of Translational Medicine, 13, 10.

Publication 2015

5-brom-4-chloro-3′-Indolyl-phosphate (BCIP) substrate

Buffers Carboxyfluorescein Fab fragments Hybridization Microrna Nitro blue tetrazolium Nucleic acid probe Phosphate Proteinase k Sheep Tissue

Corresponding Organization : University of Southern Denmark

Other organizations : Bioneer (Denmark)

Top 5 similar protocols

Variable analysis

independent variables

Proteinase-K solution pretreatment of tissue sections
Hybridization with double 6-carboxyfluorescein (FAM)-labelled Locked Nucleic Acid (LNA) miRCURY probe

dependent variables

MiRNA-126 expression (as detected by the ISH signal)

control variables

Tissue section thickness (6-μm)
Stringent washes in SSC buffers
Detection with alkaline phosphatise-conjugated sheep anti-FAM Fab fragments
4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-Indolyl-phosphate (BCIP) substrate addition
Nuclear fast red counterstaining
Automated hybridization using Tecan Freedom Evo instrument

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!