[Histonet] Rabbit-on-Rodent HRP polymer

[Melanie Black]

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.