Re: [Histonet] head kidney issues
It's endogenous biotin, Tyrone. Make your own biotin blocking kit or buy RTU kit. Also, endog. peroxidases are eliminated by 3% aq H2O2 ( saves methanol) for 10mins at RT Also, don't bother with permeabilisation.you've done all that by processing the tissue to Pwax Also, use 10% Formalin. That's what I use for my zfish Pwax IHC All suggestions: imho. Have a look in Histonet image archives for IHC Pwax sections of z.fish Best wishes Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] head kidney issues
Hello, I am doing IHC on fish tissues, in particular the head kidney. I am using DAB as chromogen. The tissue was fixed in Davidson's Fixative and processed for wax. Sections were dried in an oven overnight and then dewaxed and citrate was used for heat antigen retrieval. The sections were then permeablized with PBS-Triton and incubated in methanol with H202 for 30 min followed by 20 minutes in pH 2.3 citrate (biochemically speaking it should all be citric acid at this point) to denature any surviving endogenous peroxidase. The last step may seem a bit extreme but I was still getting signs of endogenous peroxidase activity in the head kidney sections after the methanol and still am. Now I wonder if it is something else? I found http://link.springer.com/article/10.1007/BF01003535 and saw this http://www.ncbi.nlm.nih.gov/pubmed/16242344 and now wonder if the issue isn't 5′-nucleotidase. The strong signal I am seeing has a granular appearance (macrophages?). Has anyone had similar experiences and found a solution? The former article implies using EDTA as an inhibitor. Thanks -- Tyrone Genade Orange City, Iowa tel: (+1) 712 230 4101 http://tgenade.freeshell.org Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord. To find out how to receive this FREE gift visit http://www.alpha.org. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
