[Histonet] Endothelial Cell Markers and Cell Staining

[Donna Harclerode]

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 some
work in paraffin (sort of) but work great in frozen and cells.  Human
CD34, CD105, CD106 and CD144 are all very nice markers for different
types 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 much
prefer the 24.  For some reason the 12 well plates that we have are
hydrophobic 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 S0809
for all my primary and secondary dilutions. (I add 2% normal donkey
serum 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 primary
antibody 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