AW: [Histonet] I need help with ISH

From:"Gudrun Lang"

I may be too stupid, but I don't understand your procedure.
First I don't see the denaturation-step.
What is the antigen to the primary-AB?
Is the secondary AB marked with Fluorescein?
I thought the CSA-System needs HRP on the site of the antigen and let
biotinylated Thyramid precipitate in the surrounding. What is the content of
the "Amplification reagens"?
What is your testing for?

Gudrun Lang
Biomed. Analytikerin
Akh Linz
Krankenhausstr. 9
4020 Linz

-----Ursprüngliche Nachricht-----
[] Im Auftrag von Margaryan,
Gesendet: Dienstag, 09. Oktober 2007 22:28
Betreff: [Histonet] I need help with ISH

Dear Histonetters:


I see you are from IHC/ISH Lab and I am wondering if you could help me
with the ISH problems, I am having. I am getting staining/signals in my
negative controls, in negative probes, no probes, no Primary Abs.


These are my steps. I could not get where mistake is.

I plated cells on the coverslip glass. Wait they grow for 2-3 days.
Washed RPMI with PBS, fixed in 2 different ways: 

1.	4%paraformyldehyde and store in 70% ethanol or used right away,
2.	air dry cells for an hour and fix in cold methanol for 10sec
follow by cold acetone 3x5sec.

Then using HybriProbe from Biognostik followed their protocol made

1.	rehydrate to 70% ethanol, 
2.	incubate with HybriBuffer for 3-4 h at 30C
3.	hybridize o/n at 30C
4.	quick rinse 2x in 1xSSC then for 5min
5.	quick rinse 2x in 0.1xSSC then for 15min at 40C and 45C
6.	quick wash/not wash with H2O then using kit from DAKO (CSA II,
biotin free, tyramide signal, Amplification System
7.	H2O2 block for 5 and 10 min then water wash and  wash with TRIS
Buffer with tween 
8.	Protein Block 5 and 8 min
9.	Ab primary for 15 min and 3x5 min wash
10.	Secondary Rb link(K1501) for 15 min and 3x5 min wash
11.	Amplification reagent for 15 min and 3x5 min wash
12.	Anti-fluorescein-HRP for 15 min and 3x5 min wash
13.	DAB and Counterstainig.



Please, let me know what you think.





Naira V. Margaryan, D.V.M., Ph.D.

Children's Memorial Research Center

2300 Children's Plaza, Box 222

Chicago, IL 60614-3394

Tel: 773-755-6340

Fax: 773-755-6594


For Express Mail: 

CMRC, Room C.473

2430 N. Halsted Street

Chicago, IL  60614-4314



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