I need to perform immunohistochemistry on rat free-floating brain sections. Rats were perfused with 4% paraformaldehyde. I have tried a couple antibodies but have not seen consistent results. If anyone has found an antibody for IHC that works, I would greatly appreciate knowing what company the antibody was purchased from and if any special protocols procedures were used.
Phospho-Akt (Ser473): CST #4060 is strongly recommended for both F-IHC and IHC DAB staining as well as WB.
Thank you for the information. I have tried this antibody but I have not seen consistent results for IHC. Have you personally used this antibody in your experiments?
Hi Patricia,
I've used the antibodies mentioned by Go Yoshida in western blot (http://www.sciencedirect.com/science/article/pii/S0042682214000117 OR https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection) but not sure how good it is in IHC. Are you staining tissue sections? I have used their p-mTOR too and it works fine in westerns. Are other antibodies working with your method?
-ED
Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...
Hi Patricia,
We've stained mouse skin for p-MTOR using Cell Signalling Technologies Catalogue # 2976. I believe we used 1:100 dilution overnight. These were parafin embedded and we used a pretty standard protocol (Xylene deparafinize x2, dehydrate through graded alcohols, citrate buffer boiling in microwave for antigen retrieval, peroxidase, and goat serum blocking, etc.). We recently published these data, if you want to take a closer look at our protocol and/or results: http://www.ncbi.nlm.nih.gov/pubmed/26876613
I've honestly never tried p-Akt for IHC, but this antibody works well for our westerns: Cell Signalling Technologies # 9271S (Ser 473).
Something else to consider, though, is that sometimes p-mTOR isn't a good read out of mTOR activity, due to confounding factors (like nutritional status, etc.) One thing our lab does to get around this (in Westerns, anyway) is to use downstream target levels as a measure for p-mTOR activation. We used p-4E-BP1 (CST # 9451S; negatively regulated by p-mTOR) and p-p70S6K (CST #: 9234S; upregulated by MTOR activation). I don't know if this will work for brain tissue, but it's something to consider.
Anyway, I sincerely hope this helps. Good luck in your experiments!
-Bryan
My friend used pAKT (cell signaling #4060s) for tissue slide IF, worked well.
Emmanuel: Thank you for the suggested article. Yes, other antibodies work well for my method. We have robust signaling of rpS6 phosphorylation a downstream substrate of mTOR but have had no luck with phospho-antibodies upstream of mTOR. I will try running some westerns with these antibodies for a quick check of the process. Thank you.
Bryan: Thank you for your suggested article. That's really nice work showing the p-mTOR activation. Unfortunately, my immunostaining is a bit inconsistence with fixed (4% paraformaldehyde) free-floating sections. My tissue is not paraffin embedded and I don't know if perhaps that's one reason why I have not been able to observe consistent results. I've tried a similar protocol without the initial steps (Xylene deparafinize x2, dehydrate through graded alcohol). It's possible that paraffin/slide mounted may give a different result. Also, thank you for the downstream substrate recommendation.
Hi Shiyuan,
Do you happen to know what kind of tissue they were staining?
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