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Immunohistochemistry - In Situ Hybridization: Forums

Immunoportal.com :: View topic - ISH Protocol works in mouse skin tissue
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ISH Protocol works in mouse skin tissue

 
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hopper

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Post subject: ISH Protocol works in mouse skin tissue Reply with quote
Did someone have experience in ISH in mouse skin?
I will greatly appreciate if he(she) can show me a protocol of ISH which works in mouse skin tissue.
PostThu Jun 08, 2006 2:09 pm
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Minna

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Post subject: Re: ISH Protocol works in mouse skin tissue Reply with quote
hopper wrote:
Did someone have experience in ISH in mouse skin?
I will greatly appreciate if he(she) can show me a protocol of ISH which works in mouse skin tissue.


I guess you already tried the established protocols for ISH (the one from the Anderson Lab for example - see "Protocols on Web"). If they did not work, maybe you could be a bit more specific about your problem... I'm using a slightly modified protocol and it works (at least for abundant genes).

Minna
PostFri Jun 09, 2006 4:24 am
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Minna

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Post subject: Re: ISH Protocol works in mouse skin tissue Reply with quote
I just read the Anderson Protocol again, and would really suggest you to add two things:
1. purify your probe with Roche Quick RNA Spin Columns, see picture
2. very important: pre-hybridize your sections with hybridization buffer (without probe for 1hr at hybridization temperature).

Good luck,
Minna
PostFri Jun 09, 2006 7:53 am
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hopper

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Post subject: Reply with quote
Thanks for your reply.
I have tried seveal ISH protocols but I failed. I would like to know your protocol in more detail. Cryosections or paraffin sections? Neonatal or adult skin?
And if possible, please show me some pictures of your ISH (antisence, sence probes) in mouse skin tissue.
PostFri Jun 09, 2006 8:23 am
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Minna

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Post subject: Reply with quote
I'm using cryosections (8-12um) of adult skin.
I'm actually optimizing my protocol for some time now, and might make a leap forward soon. Sorry, I don't have so much time, maybe you could post your protocol and I try to help you with it?
Minna

Last edited by Minna on Fri Jun 09, 2006 10:57 am; edited 1 time in total
PostFri Jun 09, 2006 10:26 am
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Minna

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Post subject: Reply with quote
Sorry, had to remove the pic.
Maybe later up again, but right now I'm going home...

Minna
PostFri Jun 09, 2006 10:55 am
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