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Post subject: ish mRNA, counterstain by Hematoxylin
How long should I counterstain by hematoxylin? J have 5uM sections of skin and I use DAB for visualize.
I made my first test ( I dye 10 minutes by DAB and 2 minutes by hematoxylina) and my sections made all brown :/ I know that I will have to dye DAB shorter but how long hematoxylin?????
please help mi, thanks
Wed Mar 26, 2008 6:52 pm
ole
Rank: Member
Joined: Oct 11, 2004
Posts: 855
Post subject:
Hi
A picture and a detailed protocol would help!
What hematoxylin do you use?
Usually there ( slightly dependent on DAB-type) there is rarly any effect in decreasing the incubation time (say from 10mins to eg 3-5mins).
Seems perhaps your protocol (other than the DAB and htx) needs a little tuning. A picture and a protocol might help the ISH-experts.
Ole
Thu Mar 27, 2008 11:22 am
hrn
Rank: Site Admin
Joined: Sep 27, 2004
Posts: 246
Post subject:
Magda,
If your sections comes out all brown, you should focus on your ISH protocol, not the counterstain. In case of unspsific hybridization/background, I'm afraid a reduction in DAB incubation time will not help. You have to look at probe concentration, stringency and detection method.
Agree with Ole, please share your protocol and photo if possible
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