[Histonet] cd15 and morphology on bone marrows and lymph nodes

[Andi Kappeler]

We use the rb-a-mo IgM/Biotin in stead of our usual anti-mouse Ig secondary
antibody (which is gt-a-mo Ig/Biotin). As we still stain manually (yes that
exists...!) this is no problem. Adding the rb-a-mo IgM to your usual
secondary is theoretically possible, however if your secondary is a 'multi
link' reagent (anti-mouse and anti-rabbit cocktail) then you should not add
a rb-a-mo IgM/Biotin as it will be bound by the anti-rb component of your
'multi link' reagent. The system may still work, but I would prefer to know
what I'm doing rather than to 'let something happen' inside a tube where I
don't have control. If you want to add an anti-mouse IgM component to your
'multi link', make sure it does not intefere with any of the components that
is already in there (e.g. use a gt-a-mo IgM/Biotin).

Our protocol for CD15 looks as follows (in brief):
1. Deparaffinization --> TBS
2. Pretreatment, citrate buffer, pressure cooker, 7 min at 121 C, 1 bar;
cool down, wash
3. Incubate with mo-a-CD15, clone MMA, 2.5 ug Ig/ml
4. Wash
5. Incubate with rb-a-mo IgM (DakoCytomation E 0465, 1:500)
6. Wash
7. Incubate with enzyme system (ABC/HRP, LSAB+/HRP, whatever...)
8. Wash
9. Develop with 3,3'-diaminobenzidin /H2O2
10. Rinse, counterstain, mount

Best regards,
Andi

----- Original Message ----- 
From: "Van Eyck, Deb" <deb.vaneyck <@t> phci.org>
To: "'Andi Kappeler'" <kappeler <@t> patho.unibe.ch>
Sent: Wednesday, January 26, 2005 4:20 PM
Subject: RE: [Histonet] cd15 and morphology on bone marrows and lymph nodes


> thanks very much-------for the secondary system do you just use this with
> another Dako product for example---are you using lsab or lsab +.   I know
> some people use this system and just add the IgM to the secondary--what
> specifically do you do.  this has been a real help Andi!
>
> > -----Original Message-----
> > From: Andi Kappeler [SMTP:kappeler <@t> patho.unibe.ch]
> > Sent: Wednesday, January 26, 2005 12:53 AM
> > To: Histonet; Van Eyck, Deb
> > Subject: Re: [Histonet] cd15 and morphology on bone marrows and lymph
> > nodes
> >
> > Hi Deb
> >
> > We use mo-a-CD15, clone MMA, Becton-Dickinson 347420. Working conc. is
2.5
> > ug Ig/ml, pretreatment is HIER (citrate) in pressure cooker. The
'secret'
> > with this clone (and most other commercially available CD15 mAbs, such
as
> > C3D-1, BY87,  80H5, and more) is, that it is of Ig class IgM (and not
> > IgG1,
> > IgG2a, as most monoclonals that you commonly use). While most secondary
> > antibodies show some cross-reactivity with mAbs of  Ig class IgM,  you
> > have
> > to use a mouse-IgM specific secondary antibody to get optimal results
> > (e.g.
> > DakoCytomation E 0465, rb-a-mo IgM/Biotin). This applies also to a
couple
> > of
> > other mAbs: they will work with 'regular' secondary antibodies, however,
> > they will work better with an IgM-specific secondary antibody. Therefore
> > it
> > is always useful to check the SpecSheet for the Ig-class of a given mAb.
> > If you are using a visualization system that comes from the supplier of
> > your
> > staining machine, you may have a problem, depending on the stainer. Some
> > of
> > them are obviously very reluctant to let you use secondary reagents of
> > your
> > choice... Then it is good to know, that immunos can be done manually ...
> > Hope this helps. Good luck.
> >
> > Andi Kappeler
> > Insitute of Pathology, University of Bern, Switzerland
> >
> >
> >
> > ----- Original Message ----- 
> > From: "Van Eyck, Deb" <deb.vaneyck <@t> phci.org>
> > To: <histonet <@t> lists.utsouthwestern.edu>
> > Sent: Tuesday, January 25, 2005 7:59 PM
> > Subject: [Histonet] cd15 and morphology on bone marrows and lymph nodes
> >
> >
> > >
> > >      Hi all,
> > > 1.I would really appreciate some replies to a couple of questions.
One
> > is
> > > CD15; I thought  I had a pretty good antibody going, but it is still
> > > problematic in some cases.  I would like to know what others are using
> > > consistently with heme path approval.  The same goes for kappa and
> > lambda
> > > and clonality.
> > >
> > >
> > > 2.  The other issue is handling bone marrows and lymph nodes---we have
> > > recently gotten rid of B-5 and we use NBF as our main fixative.  I
would
> > > like some replies in regards to what is the best fixation/decal
> > situation
> > > for morphology purposes (does anyone feel that they have reached the
b-5
> > > nirvana state for good morphology) and still maintain immuno
> > staining????
> > > Times and protocols and products would be great.
> > >
> > >
> > > Thanks----Deb Van Eyck-Anatomic Path
> > > Waukesha Memorial Hospital
> > > 725 American Ave.
> > > Waukesha,WI 53188
> > > 262-928-2112
> > > fax:262-928-4849
> > >
> > >
> > >
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