I wonder if you are using immunohistochemistry. Endothelial markers applied with immunohistochemistry and morphometric methods will be helpful. Without the description of your method it is hard to make any suggestions.

I should use antibody directed to CD31.

Not D-40, because this antibody is directed only to lymphatic vessels.

Yes, Sulen, we are using immunochemistry. We have tried CD34 in lung tumor cancer (NSCLC) samples and have had a very good expression of the marker. We just wondered if anybody could recommend a better method to distinguish between "normal" and "proliferative" blood vessels.

In case of immunohistochemistry it can be used the endothelial marker as CD34 or for visualisation of microvessels antibody against laminin. Very useful is to quantify microvessel by means of stereological methods and appropriate software.

CD 31 immunohistochemistry works well in our lab!

Hello Dolors, since you are asking a very specific question, here my detailled answer; this is the protocol I would use:

* Deparaffinize tissue OR use appropriate HIER buffer for simultaneous deparaffination/thermal pretreatment. For these markers I would use a EDTA based buffer at pH8

* rinse in TBS-T buffer

* 7 min Protein Block (serumfree, ideally RTU from a kit)

* blow off or if not possible rinse in TBS-T

* 30 min CD31 (clone: JC/70A at 1:50-1:200)

OR:

* 30 min CD34 (clone: QBEnd/10) at 1:200-1:400)

OR

* 30 min VEGF (rabbit polyclonal at 1:250-1:800)

* rinse in TBS-T

* 10 min Amplifier (i.e. using a modern 2-step polymer detection system like UltraVision Quanto)

* rinse in TBS-T

* 7 min Peroxidase Blocking (i.e. RTU included in the kit or homebrew 0,3% H2O2)

* rinse in TBS-T

* 10 min Polymer-HRP conjugate (i.e. UV Quanto)

* rinse in TBS-T

* 10 min DAB Quanto

* rinse in water

* 5 min counterstain with diluted MAYERS Hematoxylin

* rinse in water

* 30 sec bluing

* rinse in water

--> dehydrate slides and coverslip

Attached you will find some staining examples.

CD31 is the standard for endothelial cells.

CD34 is also frequently used for detecting endothelial cells but is less specific than CD31.

VEGF will stain growing/maturing endothelial cells and neoplastic endothelial cells that may have lost the ability to express CD31, like in Angiosarcomas.

D2-40 is generally used to detect lymphatic vessels and is not useful for the detection of endothelial cells.

Good luck !

another image: CD31

another image: VEGF

another image: CD34 in Kaposi-Sarcoma

This is fun, another image ? Here you go: VEGF in Angiosarcoma

if IHC is not a problem you can go for above mentioned procedures. or

eNOS staining can be performed. moreover massons trichrome or pentachrome movats staining visualises neoangiogenesis better than H&E or H&E with phynoxlene is better option for seeing leaky RBCs from newly formed blood vessels.

We are using immunochemistry. We have tried CD34 and CD31 and both work very well

Thaaaaanks a lot Jochen :o) And to all of you. All your answers are very helpful! We have now a plenty of choices and new ideas!!

(New ideas are still welcome....is not a message to close the subject just a thanks one!)

CD31 and CD34 are good choices.

You could also try a lectin which does not rely on immunohistochemistry and is simple. We used Ulex europeus, which is good in cobination with uimmunomethods if you want to stain for other things at the same time as vessels.

Dear Dr. Fuster,

The most reliable way to measure newly formed vessels in FFPE tissue is to use the CD105 (endoglin) antibody that reacts specifically with proliferating endothelium.

The antibodies CD31 and CD34 do not allow a distinction between quiescent and proliferating endothelium.

Agree with Frederico that pan-endothelial markers such as CD31 or CD34 would not distinguish newly-formed vessels.

However, if you quantify the smallest vascular structures (less than 20-30 micrometers squared in area, without a lumen) using these well-established markers, they usually represent neo-angiogenic sprouts.

We have traced these structures through sequential sections, and used extended focal imaging to show these are generally blind-ending and originate from larger vessels. Therefore, quantifying these structures gives a better measure of angiogenic activity (if that is what you are wanting to measure).

Another option that works well for some tumor types is to dual stain with CD31 and Dkk-1 and evaluate the ratio between the two signals. The CD105 suggestion above can also work well.

Try immunohistochemistry using heparanase-1 antibody.

Tang WQ, Hou BK. Heparanase-1 activities in the development of laser induced choroidal neovascularization. Int J Ophthalmol. 2013 Apr 18;6(2):131-5.

Great input Jochen Schuck. Dolors Fuster, are you using an antigen retrieval step after your re-hydration ?

There is no specific marker for identifying newly formed vessels in tumor tissues.

Thus, I suggest to use the classic endotehlial markers such as CD31 and CD34.

In my experience WT1 (cytoplasmic staining) is also a good marker of endothelial differentiation.

This may not be possible with your model, but I would suggst treating your animal with BrdU or EdU(invitrogen), which are thymidine analogs, and look for the CD31 positive cells that take up the compound in the time before you harvest the tissue. This will show you all of the cells that have undergone S phase of mitosis in the time between treatment with EdU/BrdU and harvest. The amount of time that you inject before you harvest the tumor will depend on your experiment. I did proliferation assays with a 2 hour window before harvest and got good incorporation in developing tissues. If you can, maybe try a time course to get a good idea of the kinetics.

Dear colleagues,

I fully agree with Dr. Netto - using antibody CD31 and CD34 together aith endoglin - CD105 seems to be very useful to be sure which vessels was newly formed. And I suggest to use the stereological method suitable for evaluation.

Hi Dr Dolores, CD31 and CD34 are good markers for blood vessels in formailin fixed tissue.

Re the lectins - which identify glycans that are stable to fixation unlike many protein epitopes - you could also try BSA-1B4 (Bandeiraea simplicifolia) also known as Griffonia lectin, and Tetragonolobus purpureus. I find they bind to different subpopulations in placenta with some overlap. See also papers by Irene Lang: Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta. Lang I, Hahn T, Dohr G, Skofitsch G, Desoye G. Cell Tissue Res. 1994 Dec;278(3):433-8

and Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta. Lang I, Hartmann M, Blaschitz A, Dohr G, Skofitsch G, Desoye G. Cell Tissue Res. 1993 Nov;274(2):211-8 which uses different antibodies. The human placenta has been likened to a tumour in many ways so you may find these techniques work for you - I hope so.