[Histonet] Re: looking for CD31 for rat tissues

[Melanie Black]

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: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of  
> Melanie
> Black
> Sent: Monday, October 19, 2009 12:29 AM
> To: histonet <@t> lists.utsouthwestern.edu
> 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.
>
>
>
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>

Melanie Black
082 469 3352

Cardiovascular Research Unit
3rd Floor; Chris Barnard Building
Medical School;
Observatory. 7925.
University of Cape Town.
South Africa.