[Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.

Adam . anonwums1 <@t> gmail.com
Thu Nov 5 10:49:22 CST 2009


More info, as Gayle rightly pointed out that I left some out.

I fixed in 10% zinc buffered formalin overnight at 4C. I decalcified in
dilute formic acid (it's called Immunocal from DeCal corporation) for 72
hours at 4C. They were embedded in paraffin. I use 5 ug / mL of the primary
antibody, and then come in with a secondary (1 ug / mL)  labeled with a
fluorophore. It works great.

I have tried it in paraffin embedded 4% PFA fixed, EDTA decalcified sections
(with or without antigen retrieval) to no avail. I think that's probably due
to the PFA and not the EDTA. I haven't tried frozens yet because paraffins
work so well.

Adam

On Thu, Nov 5, 2009 at 10:44 AM, Adam . <anonwums1 <@t> gmail.com> wrote:

> Hi Julia,
>
> Presumably, the anti-human stains mouse tissue because of protein homology
> between the human N-cadherin and mouse N-cahderin. However, I should note
> that there is evidence that this anti-human N-cadherin may cross react with
> some other cadherin on mouse tissue and thus the staining may not be
> completely specific<http://www.nature.com/nature/journal/v457/n7225/full/nature07639.html>.
> It's buried somewhere in that paper but they incubated the sections with
> excess of a different N-cadherin antibody (called MNCD2) and that didn't
> block the staining. However, that MNCD2 antibody they used for blocking
> probably also is cross reactive (I can give you references and actually seen
> that in my own hands) and really, the gold standard to show specificity for
> an antibody is to add excess N-cadherin to the antibody prior to incubation
> and showing it doesn't bind to tissue antigen anymore (alternatively, you
> could stain a knockout mouse, but those guys are lethal).
>
> IBL is a company.http://www.ibl-america.com/newproducts.html
>
> Yeah, the nomenclature for zinc is confusing. *10% zinc buffered formalin*is 10% formaldehyde (formalin) with some methanol and also zinc salts added.
> Apparently, the zinc ions bind to proteins and prevent them from
> cross-linking during fixation... if you use neutral buffered formalin, you
> get full cross linking and have to do antigen retrieval. We get the stuff
> from Fisher. The recipe you showed is *zinc fixative*, which is something
> completely different. That also prevents cross-linking. The stuff is cheap,
> so we just buy it in big jugs.
>
> Adam
>
>
> On Thu, Nov 5, 2009 at 10:22 AM, julia hough <julia.m.hough <@t> hotmail.co.uk>wrote:
>
>>  Hi Adam
>>
>> Thanks a lot for your response.
>>
>>
>> I've not been doing IHC for v. long as I've only recently graduated from
>> university, so I hope you won't mind me asking this, but how does anti-human
>> stain mouse tissue? I thought it would have to be anti-mouse?
>> Also what is the acronym IBL stand for?
>>
>> For the zinc buffered formalin is this the zinc formalin or the zinc
>> fixative as shown below, as I know there is sometimes confusion with this
>> histonet.
>>
>>
>>
>> *Zinc Fixative*
>>
>> *
>> *      Calcium Acetate ---------------------- 0.5 g
>>       Zinc Acetate -------------------------- 5.0 g
>>       Zinc Chloride -------------------------- 5.0 g
>>       0.1M Tris Buffer made above ------ 1000 ml
>>
>>
>>
>> If you’ve got any protocols that’d be lovely.
>>
>>
>>
>> Thanks again
>>
>>
>>
>> Best wishes
>>
>>
>>
>> Julia
>>
>>
>> ------------------------------
>> Date: Thu, 5 Nov 2009 10:02:02 -0600
>> Subject: Re: [Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.
>> From: anonwums1 <@t> gmail.com
>> To: julia.m.hough <@t> hotmail.co.uk
>> CC: histonet <@t> lists.utsouthwestern.edu
>>
>> I use rabbit anti-human N-Cadherin (clone YS from IBL), and it stains
>> mouse bone sections and also mouse cells by Western blot. In my hands, it
>> works great on unretrieved sections fixed with 10% zinc buffered formalin
>> fixed sections (but not neutral buffered formalin or paraformaldehyde fixed
>> sections even with antigen retrieval). I don't have it directly conjugated,
>> but with indirect immunofluorescence, the cells are quite positive so you
>> may be able to directly conjugate it yourself and get a decent signal.
>>
>> Adam
>>
>> On Thu, Nov 5, 2009 at 9:38 AM, julia hough <julia.m.hough <@t> hotmail.co.uk>wrote:
>>
>>
>>
>>
>> Dear histonetters.
>>
>>
>>
>> I am trying to perform several new IHC stains on mouse
>> tibia. However, I am having trouble finding literature on some of them. I
>> would
>> like antibodies that work on mouse tissue/bone that are preferably
>> conjugated
>> to a fluorchrome.
>>
>>
>>
>> If anyone has any potentially protocols or information where
>> I could get primary antibodies unconjugated/conjugated for the following
>> that
>> would be lovely:
>>
>>
>>
>> N-cadherin, CD146, CD144,ELF97 TRAcP.
>>
>>
>>
>> Thanks for your time.
>>
>>
>>
>> Julia
>>
>>
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