[Histonet] Reg: Beta-2 minroglobin IHC

[Liz Chlipala]

Robin

Have you tried other retrieval methods, its best to normally try to stay
away from HIER on bone but sometimes you have to use it.  If you have
not already tried other retrieval methods I would try no retrieval,
proteinase K and then trypsin or pronase, my favorite is proteinase K.
Since the samples have been decaled we have sometimes noticed in our
hands that the nuclei can  get "chewed up" a bit with enzyme retrieval
so if you notice that you may need to back off on the time of retrieval,
just as long as you do not loose staining intensity.  If this antibody
will not work with any other retrieval method then you will have to use
HIER retrieval.  Instead of retrieving at 95 or 100C or even higher I
would go with 70C for several hours (2-3) you are going to have to pilot
this. That's what we do here for any sample has a tendency to lift or
distort when retrieved (bone, skin).  You may then be able to run the
slides on a stainer but again if you see tissue detachment you are going
to have to run them by hand.

We use a good superfrost plus slide such as histobond for bone and if we
are running into problems with tissue lifting we let the unstained
slides sit for a few days even a week before we stain and we find that
that helps also.

There is a human mitochondria antibody that we have used in the past in
rat samples, but I can't remember what retrieval method works.  There is
also a human nuclear antibody which works well on frozen sections but
can be tricky on paraffin, we have not had much success with it on
paraffin sections.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Robin
Dean
Sent: Wednesday, December 08, 2010 9:20 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Reg: Beta-2 minroglobin IHC 

Hi all,

 

We are trying to use beta-2 microglobulin IHC staining to ID human cells
in
goat joints that were engrafted with human cells some time ago and
having
nothing but trouble. Tissue was decalcified in immunodecal,
formalin-fixed
paraffin-embedded tissue. Beta-2 MG antibody is a polyclonal rabbit X
human
beta-2 antibody from Acris, and we are using DAKO Envision-HRP -con.
secondary with DAB as chromogen.

IgG isotype controls and DAB alone controls look clean.

.         Goat joints are falling off the charged slides with
heat-induced
citrate buffer antigen retrieval so went to enzymatic retrieval to keep
them
on the slide.

o   Does enzymatic antigen retrieval damage this epitope?? (previous
successful uses of this antibody were with citrate antigen retrieval)

o   Any suggestions on how to keep sections on slides? Sections are kind
of
large, but pathologist wants that size.

.         Didn't get hardly any staining at 2.5 ug/ml of the beta-2MG
antibody, but at 5 ug/ml everything stains including all of goat joints.


.         Are there other or better stains that are used for identifying
human cells engrafted into other species (label uniformly most human
cells)?
I saw mention of some mitochondrial antgen antibodies but no specifics.

.         Does Beta-2  antibody label only human cells? Seems like there
are
homologous markers in other species that may label. 

Currently we aren't using any blocks except peroxidase block. Thought we
might try a CAS block to see if that will prevent sticking? To non-human
tissue.

 

Any suggestions or help with these problems will be greatly appreciated.
We
have to get the stain to work and are running out of ideas.

 

Robin 

 

Robin R. Dean, Ph.D.

Senior Scientist & Study Director

Comparative Biosciences, Inc.

786 Lucerne Dr.

Sunnyvale, CA

(408) 738-8060

robin_dean <@t> compbio.com

 

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet