We are trying to label endothelial cells post Click-IT EDU labeling, but it appears that our antigens have either been cleaved or damaged. The Invitrogen rep said that it should work, however after multiple attempts I am still not seeing my markers.
Can anyone recommend a protocol?
Thank you
She is right, proteins should not be effected unless other aspects of the reagents that come with that kit effect protein structure. I would start with looking at the ingredients of any buffers or reagents used with the EdU. If those are the problem perhaps you can revise the EdU assay using something less toxic
Thanks for your responses.
I realize that EdU labels DNA and that part of the kit works fine, it is the subsequent antibody detection that I am having trouble with.
Thanks Todd. I agree that it might be something else in kit. Perhaps the antigens are being cross-linked in the fixation step?
Very possible. What fixative are you using? What percentage and time? It could be over fixed causing the masking of antigens. You could also try changing fixatives. What about methanol?
Kit suggests to use 4%FA. The reaction based on ascorbic acid and copper to generate mild oxidation. After the reaction permeabilization is essential for immunostaining (in general, membrane labeling outside should be not, but it can help even). If this repeatedly does not works for your antigen, you should change the DNA label... or try another ab of your protein.
Sorry, i missed a word "post".
HI
If you are looking at surface antigen, it is recommended that staining should be done first followed by fixation and click detection. If you are staining only intracellular antigens, it may be better to perform the click detection before your stain as the click reaction is very stable.
Hope this helps little
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