[Histonet] Endothelial Cell Markers and Cell Staining

Andrea Hooper anh2006 <@t> med.cornell.edu
Wed Aug 2 22:07:26 CDT 2006

DAKO's anti-CD31 clone JC/70 is EXCELLENT for paraffin section staining. I am glad to share my protocol with 
anyone who is interested, just mail me offline. Both DAKO's JC70 and Pharmingen's WM59 are also excellent for 
frozen sections.

----- Original Message -----
From: Donna Harclerode <dharclerode <@t> cytoritx.com>
Date: Wednesday, August 2, 2006 9:52 pm
Subject: [Histonet] Endothelial Cell Markers and Cell Staining

> Hi Hannah
> I do lots of human endothelial staining and my best all around general
> endothelial marker is CD 31 from PharMingen, clone WM59 cat # 
> 550389.  I
> use it in frozen sections (1:40 with fluorescent secondary or Labeled
> step avidin biotin and DAB)fixed for 5 minutes in 75% acetone 25%
> ethanol (Thanks for this fix recommendation years ago Gayle 
> Callas!) or
> in cells fixed 15 minutes in 4%PFA.  I usually incubate primaries
> overnight at 4oC, but also sometimes do 1-2 hours on the counter with
> rocking.  My other endothelial markers are also from PharMingen and 
> somework in paraffin (sort of) but work great in frozen and cells.  
> HumanCD34, CD105, CD106 and CD144 are all very nice markers for 
> differenttypes of endothelial cells with the same fixation in both 
> cells and
> frozen sections. PharMingen makes excellent endothelial markers for
> mouse and rat too.  The Rat CD 31 from PharMingen cross reacts in 
> pig if
> anyone cares.  I have been staining lots of tissue culture plates and
> chamber slides with these markers double labeled with smooth muscle
> myosin or vWF and DAPI nuclei. I do not like the 12 well plates and 
> muchprefer the 24.  For some reason the 12 well plates that we have 
> arehydrophobic and I have problems covering the cells.  In a 24 
> well plate
> I only use about 100ul per well and it works nicely 
> I would not recommend most of PharMingen endothelial markers for
> paraffin sections.
> Christopher
> I noticed a couple things that might help your staining.  I would not
> use sodium azide in an antibody diluent for staining- the amount 
> may not
> hurt, but it sure does not help. I use azide in stock antibody if they
> do not come in with azide, but would not add more when making up 
> for a
> IHC (or ICC, if you prefer staining solution)  I also do not use BSA
> anymore- I have found my best results are when I use 2-5% of whatever
> normal serum from species your secondary is made in and not use 
> BSA. You
> also have no surfactant of any type to help get through the cell
> membrane. This last one would be my first guess as the problem.  There
> are also antibodies that do not work well in cells and they work great
> in paraffin sections- I have found a couple that I have rejected for
> cells and frozen that are great in paraffin sections. So much 
> depends on
> your antibody- they do not all cooperate in all formats I want them to
> work in.  
> You can make your own diluent with Tween or Triton X 100 but I highly
> recommend the prepared diluents. I think there may be some casein in
> them, but do not know the secret formula.  I use Dako diluent cat 
> S0809for all my primary and secondary dilutions. (I add 2% normal 
> donkeyserum in the primary only because all my secondaries are made 
> in donkey)
> My isotype specific secondaries are the only exception - they are all
> made in goat so I would use normal goat if I am doing multi color all
> mouse, but different isotype abs.
> I used Dako diluent when optimizing the PharMingen antibodies years 
> ago(I set up the original IHC testing and QC specs for all PharMingen
> antibodies) - I would expect other companies' diluent to work well. 
> One
> thing I have noticed is in antibodies that specifically say to "use no
> Triton in your antibody diluent" - the Dako diluent still works great.
> I have complete penetration of cells and do not damage any of the
> sensitive markers for special antibodies.  I have never found a 
> primaryantibody that works better in another homemade diluent other 
> than Dako.
> I have a protocol for plates or chamber slides I can email it if you
> want it.
> Good Luck
> Donna Harclerode, HT, (ASCP), HTL, QIHC
> Scientist / Immunohistochemistry
> Cytori Therapeutics
> 3020 Callan Rd.
> San Diego, CA 92121
> 858-458-0900 ext 5416
> fax 858 200-0945
> dharclerode <@t> cytoritx.com
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