I am doing an angiogenesis assay using a porcine carotid artery assay (it's similar to a rat aortic ring assay: Article An ex vivo angiogenesis assay utilizing commercial porcine c...
). I want to use some type of fluorescent stain for this, whether immunofluorescence, calcein AM, or something similar. However, because of using extracellular matrix (ECM) as a control substrate and the presence of other cells that will have migrated, I need to find a stain that works well for the capillaries only or my images are going to be very confusing.I was originally thinking of CD31, but it looks like it also tags to some immune cells that may also be present. Tomato lectin may work, though I am going to have to see if I can find one that will work for porcine tissues (everything I have seen suggests it works in rodents specifically). I also thought about anti-von Willebrand stains, but I believe vWF can be found in the ECM. Calcein AM will stain all living cells.
Does anyone know of a good fluorescent stain to use that is specific to porcine vascular endothelial cells, but won't tag to ECM proteins?
I take it you're using Matrigel or a similar product? If so, vWF should be a minimal component of your matrix, as Matrigel is 60% laminin, 30% collagen IV, and 8% entactin - not to mention its distribution in the matrix ought to be very different from its accumulation in endothelial Weibel-Palade bodies or in alpha granules if you have residual platelets that haven't degranulated. If your CD31 staining appears to label immune cells as well, you may be able to use antibodies against their respective cell surface markers to help distinguish them from endothelial cells. Staining for VE cadherin may also be helpful.
Re: lectin staining, any fluorophore-conjugated isolectin B4 should bind surface glycoproteins on cells from any species provided it recognizes the sugar groups present - however, this may cause you may run into even greater issues with lectin staining specificity compared to proteins themselves. If you refer to this data sheet from ThermoFisher, you'll see the number of applications for lectin staining, including macrophages, and reports that IB4 binds laminin itself https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp21410.pdf - I also have images of porcine skin co-stained with AlexaFluor 564-conjugated IB4 and anti-smooth muscle actin that illustrate the lack of IB4 specificity, which I am happy to privately share with you.
I hope this is helpful for you - good luck with your assays!
I would still recommend the CD31 which can specifically stain vessels without any background staining, you just need to find the proper concentration, also it is better to try IHC (Imminohistochemistry) with DAB, in this way you have clear image of capillaries.
Good Luck
Thanks Anthony. I'm actually using an ECM-derived hydrogel as a control, rather than Matrigel. I more or less have the full complement of proteins and related structures found in that ECM, which is why this is challenging. Thanks for the reference and explanation of lectin staining; I came across another one that lists the specific glycoproteins, and it would be a mess for me to image I suspect. I agree, that wouldn't be a good target for me to use in this case. I hadn't come across VE-cadherin, so I'll look into that too.
Thanks for the suggestion Masoumeh. This experiment takes about two weeks to run, so I worry that experimenting to find the proper concentration of anti-CD31 stain is going to be time consuming before I get the final results. The reason I am looking at immunofluorescence over immunohistochemistry is that I want to see the length and branching of the capillaries, rather than details of the capillary structure. I don't think I can do proper histology with microtome sectioning with the set-up I'm using.
One of my committee members suggested that I consider using VEGFR-2 or TIE-2 as targets. Of the two, I suspect that VEGFR-2 is going to be the better target. Any thoughts on these two?
Both TIE2 and VEGFR2 are expressed in endothelium, but there are also published data regarding their expression in immune cells, hematopoietic and stem cells including marrow-derived endothelial progenitors, as well as the function of their respective signaling axes in those populations. At the same time, these, along with CD31/PECAM and VE-Cadherin, are all used consistently as markers of endothelium. Ultimately you'll need to examine the morphology of the cells/tissues that stain positive for these in your assays to help you determine which is best for your purposes - fluorescence may complicate things due to background autofluorescence from either the collagen within the carotid sections themselves or the matrix you are using, so be certain to use appropriate controls.
DAB-based immunohistochemistry with CD31 or any of these markers, as Masoumeh mentioned, can potentially add a layer of clarity. One caution on using antibodies against porcine targets - be sure that they actually recognize proteins of the appropriate molecular weight. The best CD31 antibody I've used is a mouse monoclonal antibody clone LCI-4, first described in this paper Article Alpha-galactosyl-mediated activation of porcine endothelial ...
and which we used for fluorescent IHC in this workArticle Matrix- and plasma-derived peptides promote tissue-specific ...
I worked with pigs for much of my PhD work, and found several low-quality antibodies along the way that gave questionable staining patterns and turned out to bind several spurious entities in Western blots, none of which corresponded to known isoforms, processing stages, or glycosylated variants of the proteins in question. Good luck!
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