Fluorescence In Situ Hybridization (FISH)

( http://www.abnova.com ) - FISH is a technique used to identify and localize the presence or absence of specific DNA sequences on cells and tissues. You can use FISH probes for the detection of gene amplification, loss and translocation. Each FISH probe product has a pair of locus-specific, fluorophore-labeled probes originated from a bacterial artificial chromosome (BAC) library.

來源 : https://www.youtube.com/watch?v=dRmWZ3IUjFo

附件:

FISH.pdf (566.7 KB)

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材料準備
1.
Preparation of FISH probe

1. The following FISH probes are ready-to-use, no need of any preparation.

a. Gene FISH Probe (Cat # FGxxxx)
b. Split FISH Probe (Cat # FSxxxx)
c. Translocation FISH Probe (Cat # FTxxxx)
d. Prenatal FISH Probe (Cat # FMxxxx)
e. Made to Order FISH Probe (Ca # FAxxxx)

2. Chromosome FISH Probe (Cat # FCxxxx) and Subtelomere FISH Probe
(Cat # FExxxx) are provided in 5x concentrated format, they should be either:

a. Diluted to 1x with FISH Hybridization Buffer (Cat # U0028 or U0029) before use,
OR
b. Mixed with same category of FISH Probes (up to 5 different probes) to use, for example:

Combine 2 different probes:
1 volume of probe 1 (2 uL) + 1 volume of probe 2 (2 uL)
+ 3 volume of FISH Hybridization Buffer (6 uL)

Combine 3 different probes:
1 volume of probe 1 (2 uL) + 1 volume of probe 2 (2 uL)
+ 1 volume of probe 3 (2 uL) + 2 volume of FISH Hybridization Buffer (4 uL)
2.
Recommended filter set

The table below is a recommendation of filter set use:

Note: EX. = excitation wavelength; EM. = emission wavelength
3.
Protocol selection

Please follow an appropriate protocol below depend on the sample use, these samples include Paraffin embedded tissue (or FFPE), Frozen tissue and Metaphase spreads.

For Paraffin embedded tissue, we recommended FFPE FISH PreTreatment Kit 1(Catalog #: KA2375 or KA2691 for the pretreatment of Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections.
實驗步驟

Paraffin embedded tissue
4.

Deparaffinized

Xylene 5 min×3 Room temperature
5.

Dehydrate
100% EtOH 5 min×2 Room temperature
6.

Air dry
7.

Pre-treatment

Paraffin Pretreatment Solution 95℃ 30 min

Wash buffer (2×SSC) 5 min×2
8.

Protease treatment
Protease Solution 37℃ 10∼20min
Wash buffer (2×SSC) 5 min×2

NOTE:
*Protease Solution Add 500μl protease in 50ml protease buffer
*Protease preservation One month：4℃ Over one month：-20℃
9.

Dehydrate (Room temperature)

70% EtOH 1 min

100% EtOH 1 min
10.

Air dry
FISH protocol
11.

Mark hybridizing area

use diamond pen
12.

Apply 10μl FISH probe for 22mm x 22mm area
13.

Cover with cover glass and seal with rubber cement
14.

Denature
75℃ 5 min
Hybridization
15.

Incubation

Humidified box 37℃ 16 ~ 72 hrs
Wash procedure
16.

Remove rubber cement Slide into 2X SSC and remove cover glass
2X SSC Room temp. 5 min

2X SSC / 0.3% NP-40 73~75℃ 1-2min

2X SSC Room temp. 1 min
Counter stain
17.

Apply 10μl DAPI Solution to target area

*DAPI Paraffin embedded tissue 1500ng/ml
18.

Put on cover glass Seal with manicure
Examine