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Author Topic: Do I need to confirm my IHC result by other assay? in clearer version help pleas  (Read 3442 times)

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Offline Clear

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HI all
I have a feeling that I did not make myself clear in the last post, so I will try to make it clearer ,I am dealing with tissue culture andtreating my cultures with differrent growth factors to evaluate their effect on the phenotypic yeild of my cells, for that I cultured the cells on rounded coverslps in well plates,I usually do 3 independent experiments and in each do three biological replicants (3 coverslips) so in total I do 9 cover slips derived from the three interdependent experiments and  take randomly 5 photos from each cover slips from each condition then counts the positive cells for my different immunostaining (immunofluorescence)and compare the treated with the control , I did so for a set of 14 antibody along my study and some gave me significant results. do I need to confirm this by another assay like western or q-pcr? I really would appreciate your kind help as I am about to handle my thesis whith in few months not sure if what I have done is ok and enough
thank you very much for your help in advance  ???


Offline Cardio

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Confirmation of your results is always a good thing.

Did you look at Brdu incorportaion?

This is also a question you should ask your PI about.

Offline Clear

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Thank you very much for your reply, My PI keep telling me no need to do confirmation but my feeling that it is good to do, what assay would i go for westorn blot or Qpcr
thanks again

Offline Cardio

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I don't know what antibodies you used but both qpcr and western will work.

ITs pretty easy to just do a western with the antibodies that you have (assuming that they work) and then quantitate the bands with densotrometry.

Offline Clear

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Many thanks for your reply, will give it a go, fingers crossed :D