Hello Histonetters,
First I would like to say happy
holidays to you all, this is such a great resource to have.
Now for my question.
I'm rather vexed about this problem with staining CD4/Cd8 in mouse tumors I
hope someone can shed light on because I'm stumped.
An investigator gave me some frozen mouse tumors and spleen to do CD4 and CD8
stain on and I expected this to be easy...
After staining the spleens and tumor with CD4 and CD8 at (1ug/ml and 5ug/ml
respectively) I saw the problem, the isotype control. The isotype for these 2
Rat-mouse antibodies from BD biosciences is a Rat IgG2a.
The rat IgG2a on the mouse tumor and spleen slide were negative and I was
happy but when I looked at the other tumors Rat IgG2a slide some were very
positive. The CD4 was staining in some tumors but these tumors had background
throughout the tissue as well. Very strange I thought, some tumors no
background others lots of background..
As I'm not a tumor biologist I'll just say these tumors vary in size from
small to large, large (>1cm) and some have necrosis others do not.
So what I see is that the isotype (rat IgG2a) stained in some tumors but not
all of them and the spleen and a few small tumors are negative. I only did
Neg controls on 2 tumors one large and one small tumor with a spleen from that
tumor bearing mouse.
I first thought peroxidase or Fc binding as I don't do a protein blocking step
with this system, typically. I added a protein block (Dako) and increase
peroxidase time from 5 minute to 10 but saw no difference.
It looked like my titers were high for these tumors so I titered the antibody
out. I titered my CD4 down to 0.25ug/ml for these tumors and that did not
help the Rat IgG2a staining but the CD4 staining looked better not as dark
brown in the spleen.
So now I need to explain my staining protocol.. We use a Leica Bond RX system
which I use a DAB kit to detect with and it works great.
Marker (antibody, Rat Anti-mouse CD4) 15
Rabbit Anti-Rat
vector lab (adsorbed to mouse)
as a linking step.
20
polymer (Anti-Rabbit) 8
Peroxide block
5
DAB
10
Hematoxylin
5
I next looked at the linker step
slide #1 I did a slide with no CD4.... still had staining (background)
Slide #2 No CD4 and No linker ........No background.. clean....I think it is
the linker..
Unfortunately I'm stuck using this linker (rabbit anti-Rat , 2ug/ml) for now..
What could be going on in these tumors ??? could I block this without changing
my polymer (rabbit). If it is the linker then is it binding via Rabbit Fc or is
the hypervariable region specific for something in these tumors i.e....
Anti-Rat tumor protein?
Can I pre-complex the CD4 and linker then bind up excess anti-Rat, any one done
that???
If it is not complicated enough the linker is adsorbed to mouse.
I tried Biocares Rat-on mouse polymer but I did not see any reaction at all,
..but I haven't investigated that fully..
Sorry for the long email ..
Hope you can help...Happy holidays.
Jamie Erickson
AbbVie BioResearch Center
Pharmacology
100 Research Drive
Worcester, MA 01605
OFFICE +1 508-688-3749
EMAIL terry.me...@abbvie.com
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