FISH TECHNIQUE

Stabile Technique

11/1/2009
Medical Lab Dr Wafaa H El-Metenawi
Prepared by Ahmad Abdel-Aziz Farag

November 2009
FISH TECHNIQUE

Culture

Add 300 – 400 µl sample( bone marrow or blood) to the culture medium (RPMI and B.M.media)

in culture flask Incubate at 37ᵒC for 16 hr.

Harvest
Things to do before starting:

■ ᵒC for at least 1hr.

■ -freeze for at least 1hr.

1 Transfer the constituents of the culture flask to 15 ml falcon tube

Centrifuge at 1000 rpm for 10 min.

2 Remove the supernatant using Pasteur pipette and resuspend the pellet in 10ml

Hypotonic soln. (pre-warmed at 37ᵒC) slowly and drop by drop Incubate at RT for 30 min.

(With connuous gent l e shaki ng ever y 5 m

i n)

3 Add 1 ml pre-freezed fixative slowly and drop by drop.

(With continuous gentle shaking)

4 Centrifuge at 1000 rpm for 10 min. and

Remove the supernatant using Pasteur pipette

Prepared by Ahmad Abdel-Aziz Farag Page 2

FISH TECHNIQUE

5 Re-suspend the pellet in 10ml pre-freezed fixative

Slowly and drop by drop and incubate at RT for 10 min

(With continuous gentle shaking every 5 min)

6 Repeat the previous 2 steps 4 and 5 unl

Clear supernatant and the pellet not content RBCs

7 Save the pellet suitable fixative volume.

Prepared by Ahmad Abdel-Aziz Farag Page 3

FISH TECHNIQUE

Slide Making & Hybridization

1 Good clean the slide and cover with ethanol (70 %)

Add one drop (50 µl) on the center of the slide

Wait for dry the slides then delimitation cells location

2 Prepare the probe according to following tables

Probes (11,13 and 17) Other probes

Buffer 7 µl BCR/ABL 6 µl

H2o 2 µl X,Y 10 µl

probe 1 µl Tri 12 10 µl

Total 10 µl According to test type

3 Add adequate volume from the probes on marked area of the slide

Add the cover and remove all any air under cover

Place (UHU) around the cover then wait for dry (UHU)

4 Place the slide in Hybrid apparatus without leave any space between

The slide and Hybrid’s surface then select adequate program for each test over night

Probe (BCR/ABL),(X,Y),11,13 and 17 Program number 2

Probe (12) Program number 1

Prepared by Ahmad Abdel-Aziz Farag Page 4

 FISH TECHNIQUE

Wash Preparation

20 XSSC

■ Na Cl

■ Na Citrate

■ H2O

73ºC Wash (PH 7-7.5)

Add this soln. in bottle and place it in
water bath at 83ºC

■ H2O Place the slide after remove cover into

this bottle and shack the bol e for 4 m
i n.
■ 20 XSSC

■ NP-40

■ H2O

RT- Wash (PH 7-7.5)

Add this soln. in bottle and place it in RT

■ H2O
Transfer the slide from 73ºC wash into
■ 20 XSSC this wash and shack the bol e for 4 m
i n.

■ NP-40

■ H2O

Wait for dry slides in the dark then delimitation cells location

Prepared by Ahmad Abdel-Aziz Farag Page 5

FISH TECHNIQUE

Staining &DAPI

Add 1oµl DAPI on the same area of previous removed cover and

Place new clear cover on DAPI and remove air under cover

And add monokeer around cover then examination

Ahmad abdEl-Aziz

Prepared by Ahmad Abdel-Aziz Farag Page 6