RE: [Histonet] need help with IHC detection of human cells in mouse tissue
Thank you for your helpful suggestions! I will try some of the things you mentioned very soon.
We use Regular Cal Immuno from BBC Biochemical, and it has not been a problem at least for the GFP staining we do. I would like to try some other decal methods, but unfortunately I am quite short on time.
Do you mind if I ask, what primary antibodies have you used to detect human cells in mouse? Beta-2-microglobulin is commonly used, which is why we started with that. Are you aware of any others, ideally proteins with high uniform expression regardless of cell type? I'm a little worried about the microglobulin, because I don't know if it will be expressed in osteocytes inside the bone in particular. I had the thought of trying beta-actin, but so far I have not seen any antibody that would be specific to human vs mouse. Maybe a nuclear protein, such as histones?
From: email@example.com [mailto:firstname.lastname@example.org]
Sent: Saturday, March 20, 2004 12:01 PM
To: Garlits, John
Subject: RE: [Histonet] need help with IHC detection of human cells in mouse tissue
I routintely stain for human antigens on human xenografts in mice so I have
tried to address your concerns as best as I udnerstood your problems. Let
me know if I am missing something and I will try to help.
For routine antogens I have no problems as I often try to specifically use
rabbit pAbs to avoid mouse-on-mouse issues. When this is not an option, we
try to use directly labeled mouse primaries with biotin etc so we do not
have to use secondaries against mouse. When that is also not an option, we
use special blocking methods or kits from places such as DAKO etc (their
ARK kit is decent).
I do not routinely use decalcified tissue. When we have had to do decalc
tissue it caused problems as many of the decalc reagents obliterate
antigens (as they are HCl acid based). We now use gentler decalc methods
such as formic acid decalcifiers or EDTA decalc for such situations. This
might explain why you are not seeing the intensity or amount of stain even
on the human tissue as the antigens are being destroyed?
Companies such as Zymed and KPL sell anti-IgM antibodies absorbed against
mouse and human IgG which you could use probably directly in mouse tissue
without too much background. The amount of IgM should not that much in any
given mouse certainly compared to IgG (especially if you are working with
nude or SCID immunocompromised mice for the xenografts) so I would expect
that you don't even have to use a mouse on mouse kit ...
For the rabbit polyclonal which you are having issues with I would make
sure your secondary is absorbed against mouse serum proteins. Otherwise you
will get background. Jackson ImmunoResearch sells excellent secondaries for
this purpose and this is what I use. Buy a donkey anti-rabbit min cross to
mouse, human etc. The other option for staining mouse tissue with rabbit
antibodies is to use DAKO's Rabbit EnVision+ kit. This is an expensive kit
but is EXCELLENT and gives great intensity of stain with not that much
background and you can reduce your primary concentrations way down!
I will be glad to help as much as I can.
----- Message from "Garlits, John" on Thu, 18 Mar
2004 17:51:40 -0600 -----
Subject: [Histonet] need help with IHC detection of human cells in mouse
I was wondering if anybody could help me out. We want to look for human
cells engrafted in mice, particularly in formalin-fixed, acid decalcified,
paraffin-embedded tissue. I have so far tried two anti-human beta-2
The first I tried has been used in human/sheep transplant tissue, and I
tried it, but unfortunately it is an IgM kappa, so it failed with the
Biogenex mouse-on-mouse IHC kit, which it turns out is not made for IgM
antibodies. If anybody is aware of a good way to use a mouse IgM on both
human and mouse tissue, I'd love to know. I have looked for a good
secondary antibody for this purpose, but have had no luck so far.
The second antibody I tried is from Novocastra and distributed by Vector
Labs. It is a rabbit polyclonal to human beta-2-microglobulin. The
trouble I had was nonspecific binding in control mouse tissue. I was
thinking to use some of the Biogenex mouse-on-mouse kit reagents to help
block mouse antigens, but I do not know if this would actually work. The
second problem with this antibody was that I did not always see positive
marking in human tissue (especially osteocytes), which I thought should be
pretty well all positive since it is human tissue. Is the expression of
beta-2-microglobulin so variable?
Has anyone tried any other general human cell marker in other animals? I
did a quick search for beta actin, but it seems most of those cross-react
John Garlits, M.S.
Senior Research Technician
Hematology Oncology Division
Experimental Hematology Department
St. Jude Children's Research Hospital
332 N Lauderdale
Memphis, TN 38108
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