My iNOS stainings are not working. I am using the Santa Cruz Biotech anti NOS2 antibody (sc-7271), but I have only been getting non-specific stainings. Has anyone ever used this antibody on PFA-fixed brain tissue?
During formalin fixation methylene bridges formed which cross-linked proteins and mask antigenic sites.
Heat-induced antigen retrieval methods are the best ones to break these methylene bridges and expose antigenic sites, to bind with the antibodies.
You can use a pressure cooker, water bath, or microwave any of the methods.
Hello Saulo Cruz
It could be the problem of auto fluorescence of the brain tissue. Auto fluorescence can present a challenge when working with brain or neural tissues due to the variety of cell types and the accumulation of lipofuscins that tend to stick around in neural cells. Lipofuscin, known as an aging pigment, accumulates in neural tissues and causes auto fluorescence.
Intact tissues, particularly neural tissue, also have lipid bilayers that cause light scattering and auto fluorescence. Moreover, formaldehyde fixation also causes background fluorescence. All of these could make it practically impossible to work with brain tissue.
You can significantly reduce auto fluorescence by treating the sections with sodium borohydride (NaBH4) followed by cupric sulfate (CuSO4) in ammonium acetate buffer. For more information on the protocol, you can refer to the article attached below.
Article Autofluorescent cells in rat brain can be convincing imposto...
I hope this is helpful.
Best.
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