In situ hybridization detection

NegotiableUpdate on 05/06

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Overview

The basic principle of in situ hybridization detection is to use complementary base sequences between single strands of nucleic acid molecules to pair radioactive or non radioactive exogenous nucleic acids (i.e. probes) with the DNA or RNA to be tested on tissues, cells, or chromosomes, forming specific nucleic acid hybridization molecules. Through certain detection methods, the position of the nucleic acid to be tested on tissues, cells, or chromosomes is displayed.

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FISH experimental method and steps

One Probe denaturation

Incubate the probe in a 75 ° C constant temperature water bath for 5 minutes, then immediately place it at 0 ° C for 5-10 minutes to denature the double stranded DNA probe.

II Specimen denaturation

① Bake the prepared 5-8um glass slide specimens in a 50oC incubator for 2-3 hours. (Giemsa stained specimens need to be pre cooled in the fixative before baking).

② Take out the glass slide specimen and immerse it in a denaturing solution of 70% formamide/2 × SSC at 70-75 ° C for 2-3 minutes.

③ Immediately dehydrate the specimens in a series of 70% volume fraction, 90% volume fraction, and 100% volume fraction ice ethanol for 5 minutes each time, followed by air drying.

III hybridization

Drop 10 μ L of denatured or pre annealed DNA probes onto denatured and dehydrated glass slide specimens, cover with an 18 × 18 coverslip, seal with Parafilm, and hybridize overnight (approximately 15-17 hours) at 37 ° C in a moist cassette. Due to the limited amount of hybridization solution, high hybridization temperature, and long duration, this process is carried out in a wet box to maintain the moist state of the specimen.

4 elute

This step helps to remove non-specific binding probes, thereby reducing the background.

(1) On the day after hybridization, remove the specimen from the 37oC incubator and gently remove the cover glass with a blade.

(2) Wash the hybridized slide specimens three times in a preheated volume fraction of 50% formamide/2 × SSC at 42-50 ° C for 5 minutes each time.

(3) Wash 3 times in 1 × SSC preheated to 42-50oC for 5 minutes each time.

(4) Wash the slide specimen lightly in 2 x SSC at room temperature.

(5) Remove the glass slide and let it dry naturally.

(6) Take 200 μ L of counterstaining solution (PI/anti fade or DAPI/anti fade staining solution) and drop it onto a glass slide specimen, then cover it with a cover glass.

Five Fluorescence microscopy observation of FISH results