I used to fibroblast as a negative control for Oct4 immnostaining but I observed that my fibroblast was stained by Oct4 primary antibody. I also used to the other negative control with only secondary antibody and there was no background staining. In my opinion my fibroblasts nucleus were stained really specific by Oct4 primary antibody. But I couldn't find any reason to explain it. Is there anybody to help me and give me some advise?
Thanks advance
Did you cultured your fibroblast with FGF2 (fibroblast growth factor)?
If, the positiv result for Oct4 is normal. You can read it here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925031
Dear Prof. Dr. Dittmann, first of all thank you so much your kindly answer and suggestion with a paper.
No I didint. I used to common fibroblast media (DMEM, 10% FBS, 1% non-essansial aminoacid) with 2% gelatine coating. I will read the paper that you send. ALso my treated cells expressed Oct4 but more than fibroblast. Can i say my cells became pluripotent cell like cell? Also there is some specific straining with SSEA4 but not in fibroblast only in treated cells?
Usually you will not get positive staining for Oct4 in fibroblast. This may be due to non-specific staining. Please check your control cell without primary antibody to rule out the non-specific staining. I do agree that there will be some Oct4 baseline mRNA expression but not the protein expression.
NO, you should not see Oct4 expression in fibroblast or most any adult tissue other than the germline. Antibodies cross react and these can also be specific cross reactions, i.e. your controls are negative. If you have any doubts try to verify with RT-PCR or a northern blot for mRNA. But I actually did the first expression analyses of Oct4, with Hans Schoeler, and we only saw Oct4 in ES cells, the epiblast, and in the germ cells. There is no Oct4 in any adult tissue and that includes adult stem cells.
Please try with Western blot analysis. I usually do mRNA expression followed by Western and immunostaining. I never get Oct4 expression in any of these methods in the fibroblast.
"Can i say my cells became pluripotent cell like cell?"
You should also stain for Nanog, Tra-1-60 and so on.
Oct 4 is allways use also for iPS cells, but as Gregory mentioned and thats no new finding, in adult stem cells there are other findings:
http://www.nature.com/news/2007/071010/full/449647a.html
Thank you for all your kindly response.
I cheched my negative control for only secondary antibody and there is nothing as this. As you suggest i will try western and RT-PCR.
My treated cells have been stained with SSEA4 and Oct4. ( ı havent got Nanog,Sox primary antibody). But some fibroblasts are stained with only Oct4 not with SSEA. I am adding to my photo with Oct4 staining.
Thank you all again..
Try change the primary antibody. Unspecific staining probably caused by Oct4 splice variants..
http://www.ncbi.nlm.nih.gov/pubmed/18787205
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