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

From:"Andi Kappeler"

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" 
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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