Anti-CD62E antibody [1.2B6] (ab6630)

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Effectively all of the products mentioned are suitable, and therefore are guaranteed for IHC in frozen sections in the species stated on their datasheets.

However, instead of using PFA as a fixative, I would better suggest using acetone, methanol or ethanol at -20°C for 30 min, so the additional antigen retrieval step can be skipped. If, by the tissue’s nature PFA is required as a fixative, antigen retrieval is recommended to allow the antibody reach the epitope.

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I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us.

You are quite correct. As CD62E is a single pass membrane bound protein, there should be no need to permeabilze the cells for an immunocytochemical stain.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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I have received feedback from the source of ab6630 and ab19264 and would like to share their feedback. Unfortunately I am still awaiting feedback regarding ab2213. For ab6630 we would suggest a working dilution of 10ug/ml. For ab19264 we recommend to use 0.5 - 1µg antibody for 106 cells(I am still awaiting feedback from the source of ab2213 to find out the recommended dilution). We think it is indeed very likely that the "cell dissociation solution" is either removing or damaging these cell adhesion molecules on HUVECs. It is not clear what this solution is, but if it is trypsin or something similar to trypsin, this could be the source of your problem. HUVECs should be positive for ICAM-1 and VCAM-1, but the expression of those proteins can be low if the cells aren't stimulated by inflammatory mediators. Furthermore, we do not know if HUVECs express ELAM-1 unless stimulated in the appropriate manner. The following reference may help: Schatz F, Soderland C, Hendricks-Munoz KD, Gerrets RP, Lockwood CJ. Human endometrial endothelial cells: isolation, characterization, and inflammatory-mediated expression of tissue factor and type 1 plasminogen activator inhibitor. Biol Reprod. 2000 Mar;62(3):691-7. I would like to suggest that you try mixing the primary antibodies with your cells at room temp instead of on ice and as soon as I find out the recommended dilution for ab2213 I will let you know, thank you for your patience.

The antibody will recognise the native form of the epitope thus Westerns must be conducted in non-reducing conditions (i.e. without DTT/ beta-mercaptoethanol

The antibody has been characterised extensively and published in: J Immunol 1990 Apr 1;144(7):2558-65 A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. Wellicome SM, Thornhill MH, Pitzalis C, Thomas DS, Lanchbury JS, Panayi GS, Haskard DO.
and
J Immunol 1990 Aug 1;145(3):865-72 IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma. Thornhill MH, Haskard DO.
It's use in immunohistochemstry has been described in: Am J Physiol 1994 Jan;266(1 Pt 2):H278- Characterization of E-selectin expression in vivo with use of a radiolabeled monoclonal antibody. Keelan ET, Licence ST, Peters AM, Binns RM, Haskard DO.