[Histonet] IHC after EDTA decalcifying

[koellingr <@t> comcast.net]

Hi Colleen, 
glad you are also using a mouse control and looking for Ki-67 is reasonable with caution. Here is my take on this although certainly there are differing opinions from mine and no lack of them. I agree with what Ronnie said and here is my personal input. I would always put a piece of spleen or lymph node right in cassette with 1 femur to be sure it (control target) was fixed, decaled and treated the same as femur target. Or another tissue depending on target. For something like Ki-67, would use a cx of intestine (you can see the proliferating cells in crypts and they lose positivity when they are pushed up the villus-or a LN or spleen that I was sure had cells undergoing proliferation). IHC protocol was nothing special at all, hour on bench in primary. Or ON at 4 degrees. Normal IHC. While you can see proliferating cells in hematopoeitic portion of femur I'd use caution. Perhaps me being originally from Missouri. The way I operate, I'm skeptical of "positive" staining if it doesn't make sense rationally or biologically. I used to see, and I still see after checking, pictures of IHC stains from companies on web. Just saw one. A picture of normal skin stained with Ki-67 but I'm seeing positive nuclei all over the place (not only where I'd expect it but including all through the dermis and deep dermis in what are probably fibroblasts). How can that be? Ki-67, if what I've learned of it is true, is present in G1 and S and G2 and mitosis but ABSENT in G(0) when cells are quiescent. All those cells I've seen from a company website picture can't possibly all be proliferating. Otherwise its a tumor and not normal skin. So that is why I'd use a control which had to have proliferating and quiescent cells for something like Ki-67. Or I wouldn't believe test staining until I believed my control. There are proliferating cells in hematopoietic portion of femur, but also can be G(0) cells. Nothing special about the IHC. But that is just one persons opinion. 


Ray 
UW Seattle 

----- Original Message -----
From: "Colleen Forster" <cforster <@t> umn.edu> 
To: "Histonet" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Thursday, August 15, 2013 4:58:35 PM 
Subject: [Histonet] IHC after EDTA decalcifying 

Hello fellow histonetters, 

Can someone who has worked with decalcifyied bone tell me if EDTA 
interferes with IHC staining? I was under the impression it did not but 
cannot get staining in mouse femurs that I have decaled in EDTA. I have 
used both the steamer and the decloaker for retrieval and in the human 
tonsil the stain is great. 

Any suggestions...... 

Thanks in advance! 

Colleen Forster 
U of MN 

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