Hi Patsy
Yes it works very well. The antibody is cat # RDI-RTCD31-3A12 from
Fitzgerald. Yes it is zinc fixed. You use alk phos instead of DAB.
See pic.
Zinc Fixative (JB Fixative or ZSF)
0.1M Tris Buffer, pH 7.4
Tris Base -------------------------------- 12.1 g (TRIZMA)
1N HCL ----------------------------------- 81.5 ml
Distilled water -------------------------- 900 ml
Mix to dissolve. Adjust pH to 7.4
Zinc Fixative
Calcium Acetate ---------------------- 0.5 g
Zinc Acetate -------------------------- 5.0 g
Zinc Chloride -------------------------- 5.0 g
0.1M Tris Buffer made above ------ 1000 ml
Mix to dissolve. The final pH will be approximately 6.5-7.0. Do not
readjust the pH, as this will cause the zinc to come out of solution.
Store Zinc Fixative at room temperature. Fix tissues for 24 to 48
hours. Fixation longer than 48 hours may make the tissue brittle and
difficult to cut.

Good luck
Melanie.
On 19 Oct 2009, at 2:57 PM, Patsy Ruegg wrote:
Melanie,
I am very interested in your CD31 protocol, does it work on rat
tissue, do
you have to fix in zinc fixative?
Cheers,
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: [email protected]
[mailto:[email protected]] On Behalf Of
Melanie
Black
Sent: Monday, October 19, 2009 12:29 AM
To: [email protected]
Subject: SPAM-LOW: [Histonet] Rabbit-on-Rodent HRP polymer
Hi Jen
In reply to your question on the Biocare Rabbit-on rodent HRP, you
are correct. I include the protocol I use, the only difference is
mine is Alp phos linked instead of HRP. I use rat tissue and CD 31
antibody (from Fitzgerald) that specifically requires zinc fixed
tissue. For the zinc fixation, I use exactly the same formula that
Gayle Callis sent out.
CD 31 Alk Phos Method for Zinc fixed Tissue.
Biocare Medical Kit.
Dewax slides in xylene.
Take slides thru alcohol to water, and into tris buffer.
Incubate with Rodent Block R (RBR962H) from Biocare Medical, for 30
mins.
Wash in TBS X 2.
Incubate with Primary antibody for 30 mins.(1:50) - diluted in 1% BSA
in PBS. Anti Rat CD 31 from Fitzgerald.
Wash in TBS X 2.
Incubate in Mouse-on-Rat AP-Polymer (MRT623H), Biocare Medical, for
20 mins.
Wash in TBS X 2.
Add a drop of NBT(DAKO - K0598), and check for colour development.
(YOU WILL USE DAB)
Rinse in water, dehydrate clear and mount.
NB: Sections must be cut onto UNCOATED slides, and incubated on a 60
degree hot plate for 30 mins, followed by overnight incubation at 37
degrees.
Hope this helps
Regards
Melanie.
Hi All,
I recently purchased a rabbit-on-rodent HRP polymer from Biocare.
I will be using this on mouse tissue, using a rabbit polyclonal CD3
as my primary. I just wanted to clarify a couple things about the
protocol before I try it out tomorrow and was wondering if anyone
could offer some assistance. First of all, I should aske if anyone
has a protocol for this? What I really would like to make sure is
that I will not have to use a label after the rabbit-on-rodent
secondary ab. I believe that I will just have to apply my blocking
agent, avidin/biotin blocks, primary antibody, secondary anitbody,
followed by chromogen, right? My blocking agent is made up of casein
and some other proprietary agents in a phosphate buffer (it is non-
serum) so I was told that should work fine. Does this sound
correct? Thanks in advance! I haven't been able to get through to
tech support all day, so hopefully you will have some answers for me!
Jen
Melanie Black
082 469 3352
Cardiovascular Research Unit
3rd Floor; Chris Barnard Building
Medical School;
Observatory. 7925.
University of Cape Town.
South Africa.
_______________________________________________
Histonet mailing list
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http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Melanie Black
082 469 3352
Cardiovascular Research Unit
3rd Floor; Chris Barnard Building
Medical School;
Observatory. 7925.
University of Cape Town.
South Africa.
_______________________________________________
Histonet mailing list
[email protected]
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
