HER-2/neu FISH protocol

From:Bob Gasparini <BGasparini@USLABS.net>

Hello all,

I have gotton a couple of requests for the Ventana HER-2/neu FISH protocol.
Currently it is sold as an automated procedure for use on the Ventana
BenchMark system, however, below is the MANUAL protocol we worked out using
the Ventana HER2 reagents that come in the dispensers.

Feel free to e-mail if you have any questions.

Bob
 
Manual HER-2/neu FISH technique using Ventana's automated HER2 reagents

1.	Deparaffinize 3 times Xylene 2 min each, 2 times 100% EtOH 2 min
each, airdry
	
2.	Digest 40 ml Reaction Buffer + 4 dispenses Protease 1; place in
glass Coplin jar 50* C 40min
	
3.	Rinse 3 times 2X SSC, 2 min each with constant shaking
	
4.	Dehydrate (1) 95%, (2) 100% EtOH 1 min each, air dry
	
5.	Dispense 1 dispense (100ul) HER-2/neu probe and apply 24x50 mm
coverglass
	
6.	Denature on 75* C slide warmer 15 min
	
7.	Hybridize Place slide on rack elevating slide over water in 39* C
H2O bath 4-15 hrs
	
8.	Soak off coverglass in 2X SSC at room temperature
	
9.	Stringency/Post wash 72* C 0.5X SSC for 5 min
	
10.	Rinse 3 times 2X SSC, 2 min each with constant shaking
	
11.	Detect by placing slides on rack in 37* C H2O bath without draining
fluid and apply 1 drop FITC AntiBiotin to each slide and incubate 20 min in
water bath
	
12.	Rinse 3 times 2X SSC, 2 min each with constant shaking
	
13.	Amplify signal by placing slides on rack in 37* C H2O bath without
draining fluid and apply 1 drop AntiMouseFITC to each slide and incubate 20
min
	
14.	Rinse 3 times 2X SSC, 2 min each with constant shaking
	
15.	Counterstain with 20 m l PI/Vectashield, apply coverglass, blot one
edge with Kimwipe
	
16.	View under fluorescence microscope (100W HBO bulb) with either
FITC/TR or FITC/PI dual band filter.






Robert Gasparini
Director, Genetics
US Labs
16255 Laguna Canyon Road
Irvine, CA 92618
(888) 450-0145 x129
(949) 450-0146 fax 




<< Previous Message | Next Message >>