I agree with Darius and Sylviane, but it depends on what you want to study. If it's the total distribution of the receptor or the membrane distribution or the signalling or the traffic...

Hey Sivasankar,

the main part of E-cadherin (CD324) is localized at the surface of the cell. Thus, permeabilization is not required (and may be disadvantageous) for immunocytochemistry.. However, as usual it depends on the antibody and epitope, so you have to check it carefully using appropriate controls.

Cheers

Darius

I agree with Darius. Check the epitope recognized by your antibody (is it inside or outside the cells). Also define if you are interested by the intracellular pattern of localization as the cytoplasmic domain can migrate into the nucleus.

NO, I don't agree with those guys, I think you still need to permeabilization though E-cadherin is membrane protein, it may redistributed to cytoplasma, you need to label the whole protein distribution, not only membrane one, right?

I agree with Darius and Sylviane, but it depends on what you want to study. If it's the total distribution of the receptor or the membrane distribution or the signalling or the traffic...

hi guys, thanks for the reply. I want to study the integrity of cells junction, so membrane staining is enough for me. Am using rabbit mAb, generated against synthetic peptide corresponding to sequence of Pro780 of human E-cadherin. I dont know where it is present? i.e on the surface or on cytoplasmic side.

I'm agree with Romain. But I prefer use Triton X100 to permeabilize the cells (Concentration 1/100)

hi georg, ya i found the same using uniprot. i am thought of using 0.1% tir-x 100. and will try methanol fixation and 4%PAF fixation. thanks for the ans, and you confirmed me that am in right path. thanks

Pleased to help. ;)

Hi, Sivasankar:

Thank you for the question. I really think that if your protein is membrain protein you don´t need permeabilization. However in he case be a domain intracytoplasmatic you need research which is the best fixation using all the control to not obtain inespecificity. You need also know if your protein in soluble in triton X-100 because in that case you have to be very carefull using tritón because you can lost the detection of your epitope. You can also try using parformalaldehide and glutaraldehide (very low concentration of glutaraldehide) avoiding obtain inespecificity.

Easy to check your epitope when you stain mildly trypsinized cells - in case you have an extracellular epitope you will lose staining. in any other cases MetOH fixation works best for e-cad stainings in epithelial cells (also permeabilizes membrane).