[Histonet] need help with IHC detection of human cells in mouse tissue

[Garlits, John]

Dear Andrea,

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?

Thanks again,

John


		-----Original Message-----
		From:	andreah <@t> imclone.com [mailto:andreah <@t> imclone.com]
		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





		Dear John,

		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.

		Cheers!
		Andrea

		----- Message from "Garlits, John" <John.Garlits <@t> stjude.org> on Thu, 18 Mar
		2004 17:51:40 -0600 -----

		To:   <histonet <@t> lists.utsouthwestern.edu>
		Subject:    [Histonet] need help with IHC detection of human cells in mouse
		tissue

		Hi,

		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
		microglobulin antibodies.

		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
		with mouse.

		Thank you!

		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