Microwave-stimulated antigen retrieval is pH and temperature dependent.
The effects of the pH of solutions used in microwave-stimulated antigen retrieval were investigated for two antibodies commonly used in neurosciences, MAP-2 and SMI-32. A prolonged fixation time (longer than 1 week) prevents good immunohistochemical staining of these and many other antigens in paraffin and vibratome sections. On human brain tissue stored for 17 months in formaldehyde, the effects of different solutions during microwave treatment were compared, i.e., 4% aluminum chloride (pH 2.75), 0.1 N hydrochloric acid (pH 0.98), and citrate buffers (pH 2.5, 4.5, 6.0), together with the influence of temperature and microwave irradiation. We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied. The SMI-32 staining results are optimal with citrate buffer at pH 2.5 and 2-hr irradiation at 90 degrees C, whereas MAP-2 staining results are optimal with citrate buffer at pH 4.5 and 10-min full-power irradiation. In the other conditions the staining is less intensive or less homogeneously distributed over the section.
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Netherlands Institute for Brain Research, Graduate School Neurosciences Amsterdam, Amsterdam.
Uylings
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D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D001923:Brain Chemistry; D002675:Child, Preschool; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D008872:Microwaves; D016900:Neurofilament Proteins / Q000032:analysis* / Q000276:immunology; D017397:Prefrontal Cortex / Q000737:chemistry; D013696:Temperature; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D000941:Antigens; D016900:Neurofilament Proteins