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How important are denaturation and hybridization temperature in ISH?
Correct denaturation and hybridization temperatures are paramount for successful ISH. Regardless of the probe or target being RNA or DNA,…
Tue, Jul 18, 2023
What are ISH probes typically labeled with?
Common labels include biotin, digoxigenin, and fluorochromes. Biotin is detected with streptavidin- conjugated enzymes followed by an enzymatic colorimetric reaction.…
Tue, Jul 18, 2023
How are ISH probes labeled?
There are three main techniques for probe labeling. Labeled probes can be generated by incorporating labeled nucleotides during the synthesis…
Tue, Jul 18, 2023
What are the advantages of digoxigenin labeling?
For chromogenic ISH, Digoxigenin, compared to biotin, offers the advantage of lower background, higher specificity. Is also possible to combine…
Tue, Jul 18, 2023
How to cover up the sections during hybridization in ISH protocols?
The most common method is to cover tissue sections on glass slides in the hybridization solution with a glass cover…
Tue, Jul 18, 2023
Why are Aqua 431 dUTP-labeled probes much harder to detect?
There are two aspects making Aqua-labeled probes harder to detect: First, the aqua fluorophore (DEAC – 7-Diethylaminocoumarin-3-carboxylic acid) has a…
Tue, Jul 18, 2023
Why should post-hybridization washes be carried out?
Post-hybridization washing after ISH will reduce non-specific signal by removing probes that are lightly bound to off-target sequences. Most frequently,…
Tue, Jul 18, 2023
RNA Probe Design and Generation – How are RNA probes prepared?
RNA probes are usually prepared with biotin or specific fluorophore by in vitro transcription. High specific activity RNA probes or riboprobes…
Tue, Jul 18, 2023
How big are ISH probes?
There is no defined answer. Depending on the tissue specimen, target type, and sequence, ISH probes might vary from below…
Tue, Jul 18, 2023
Why are the Gold 525 dUTPs bleeding through in the red channel?
If an epifluorescence microscope is used for visualization, the incorporated dUTPs should be matched to the filters equipped in the…
Tue, Jul 18, 2023