Hi!
According to our knowledge, osteopontin (opn) is an extracellular protein. Yet when we discussed the staining image today, some colleagues insisted that opn is intracellullar, surrounding the nucleus and unlike the image showing the thread-like shape. In addition, in some of our image, even the actin (green) and opn (red) images of same location are all thread-like, the image from different fluorescent filters are not absolutely corresponding and overlapping. Could you please tell us how exactly should opn distribute under fluorescent microscopy?
Thanks a lot!
Regards!
Thanks! Yeah we also check with colleagues from other lab, but they say the location of opn is depend on the primary antibody. For the one we used (Abcam ab8448), it should be bright area around the nuclei, while what we see is thread-like, quite the same as the actin channel (we're doing msc for osteogenesis). Somebody said it might be the actin trapping the secondary antibody for opn, but I believe it's not, as such thread-like shape does not occur on our - control sample's image. If it's noise by the actin, how can the - control get avoid of this? We also check the product page on abcam, their image on MF127 cell are quite thread-like, overlapping with actin, while their image of the newly developed skeleton section are just bright spot surronding cells. We're really confused Mohamed Elkablawy
could you pleas tell us whether the opn should be in similar shape and distribution as actin?
sorry our images previously uploaded in the 3rd answer are raw without color balance condition, here are the images. Could you please tell us whether our opn is real opn other than the actin noise?
Thank you very much!
Regards! Mohamed Elkablawy
I gave up, the images can't display correctly on the website, pleas download and open them in imageJ. Thanks!
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