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

Thu Nov 5 12:26:04 CST 2009

Adam, 

Thank you telling how you prepared bone for N-Cadherin IHC.  I am not sure
there will be success using any fixative with formaldehyde in the recipe or
Paraformaldehyde with the murine CD146, CD144 though.  I suggest Julia go to
BD Biosciences, SEROTEC, and eBiosciences websites to pick up the IHC
applications and fixation needs for these murine CD markers, often picky and
not stainable after zinc formalin, NBF or PFA.  Start with one company, and
if they don't have the antibody, then try the next.  Company technical data
sheets should tell her if these antibodies will stain formalin fixation
and/or only on frozen sections of bone, unfixed.  If aldehyde fixatives
work, she may have to protect the antigens further by using EDTA instead of
an acid decalcifier. 

Molecular Probes has the kits and protocols available for ELF 97, also
Chemicon - and their company technical services will help on line. I believe
the TRAcP was recently and has been discussed often in the past on Histonet.
A search Histonet Archives should provide more answers. 

I wish her luck on immunostaining success, often tough when you can't stain
for all the antibodies and possibly perform ELF 97 or TRAcP on a single
sample if fixation and/or decalcification compromise(s) the results.   

Gayle M. Callis
HTL/HT/MT(ASCP) 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Adam .
Sent: Thursday, November 05, 2009 9:49 AM
To: julia hough; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.

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.ht
ml>.
> 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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