I am studying lipids in human diseased tissues. My tissues are formalin fixed, then they undergo a lipid pre treatment utilizing linoleic acid and chromic acid (Tracy & Walia, 2002), paraffin embedded, deparrafinized, then stained via immunofluorescence (IF). When I stain via IF with my primary rabbit polyclonal and goat-anti-rabbit 488 secondary and my control with rabbit serum IgG and goat-anti-rabbit secondary, the tissues look the same due to unspecific binding. I tried this on non-pretreated tissue and there is no issue, so we concluded the unspecific binding is due to the lipid pretreatment. Does anyone know what I can do to reduce the unspecific binding. *NOTE: the lipid pretreatment is essential and already done to my tissues.
1. Is there a monoclonal version of this particular antibody? It may provide you with less non-specific staining?
2. If testing another antibody isn't an option, you could always try pre-incubating your antibody in some lipid pre-treated tissue (that you're not going to use) to reduce the non-specific binding. Next, you'll collect that used antibody and incubate the tissue-of-interest with this "pre-incubated" antibody in hopes that you've removed some of the non-specific binding.
Unfortunately (in my opinion) with this kind of treatment (formalin fixation, linoleic acid and chromic acid pretreatment) it is not possible to get relialbe immunohistochemical results as als antigens in the tissue are severely affected.
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