Fluorescent in situ hybridization,FISH is a powerful technique for detecting RNA or DNA sequences in cells, tissues, and tumors. FISH provides a unique link among the studies of cell biology, cytogenetics, and molecular genetics.

Fluorescent in situ hybridization is a technique in which single-stranded nucleic acids (usually DNA but RNA may also be used) are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences. Through nucleic acid hybridization, the degree of sequence identity can be determined, and specific sequences can be detected and located on a given chromosome.

The procedures BIOLINK provided are as follows:

Denature the probe and the sample

probe preparation

Heat for 5min at 75 deg C in a water bath ,and then instantly place to a fresh jar at 0 deg C for 1~2min to denature the dsDNA .

sample preparation

Bake the slides for 2~3 hours at 50 deg C in the incubator

Place denaturant solution (70% formamide/ 2xSSC) in 70~75 deg C water bath inside coplin jar,and immerse the slides for 2~3min Dehydrate in 70%, 85% and 100% ethanol for 2 min. each. Airdry.

In- situ hybridization

Applied 10uL denatured probe and cover with a glass and seal carefully with a Parafilm.

Place slides in a prewarmed humidified box (wrapped in metal foil to protect against light) and incubate overnight at 37deg C

Washes

Remove cover glass and immediately place into wash tank(prewarmed to 42~50 deg C) with 40% formamide/ 2xSSC,wash three times for 5min each

Wash three times in a wash tank prewarmed to 42~50 deg C with 1xSSC for 5 min each

Wash the slides gently at room temperature with 1xSSC

Notebook: This step is intended to prevent the non-specific hybridization and minimize the background.

Signal amplification

1 Add 150mL blocking solution I to the hybridization area and cover with a film ,then incubate at 37 deg C for 20 min

2 Remove the film，and add 150mL avidin-FITC to the sample，then cover with a film and incubate at 37 deg C for 40 min。
3 Place the sample into the wash solution prewarmed to 42~50 deg C, and wash three times for 5min each

4 Apply 150mL blocking solution II to the hybridization areat,hen cover with a film and incubate at 37 deg C for 20 min。

5 Remove the film，and add 150mL anti-avidin to the sample，then cover with a fresh film and incubate at 37 deg C for 40 min
6 Place the sample into the fresh wash solution prewarmed to 42~50 deg C, and wash three times for 5min each
7 redo the step1，2,3,and then wash the slides  at room temperature with 2xSSC
8 Airdry slides in darkness
9 Apply 200mL PI/antifade to the slides，and cover with a glass。

Detection

Seal the slides with  Parafilm

Examine slides on a fluorescence microscope