[Histonet] immunohistochemistry

[anh2006 <@t> med.cornell.edu]

Does the marrow stay on but the cortical bone comes off?
Are they paraffin sections?
What is your decal method?
Do you use coated slides?
Do you "melt" your sections onto the slides before deparaffinization?

Even so, I suspect the reason is your antigen retrieval solution is too hot and too vigorous. Lower the temp slightly and reduce the time. There is no need to boil for most antibodies. I do 95 deg C for 10 min with a 10 min cool down. Sometimes in super fresh sections cortical bone partially comes off, but marrow nd trabecular bone stays so I can get some information.

Good luck!

-----Original Message-----
From: Hatem Salim <dr.hatemsaied <@t> yahoo.com>

Date: Thu, 04 Jun 2009 20:01:50 
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] immunohistochemistry


HI 
 I am Research assistant at Physiology department of Michigan state university. I am using the (ß-Catenin Antibody (Carboxy-terminal Antigen) #9587 for IHC on mice femur sections. I have used this antigen unmasking method: For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
The problem is that bone detachment always occurs so I wonder if there is a method of retrieval that prevents bone detachment.
> Waiting to hear from you soon.
>  Thank you for your consideration
> Best wishes
> Hatem Salim




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