An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative.
In the last 5 years the microwave oven has become a widely accepted apparatus for retrieving all kinds of antigens that are masked by prolonged formaldehyde fixation. However, it has so far not been possible to obtain good results for all the antigens we were interested in with just one method. This study offers a solution for this problem. We tested Tris buffered saline (TBS) pH 8-10 in order to see whether a microwave (MW) pretreatment in a solution with a high pH works on the antigens that have our particular interest: MAP-2, non-phosphorylated part of the neurofilament (SMI-32, SMI-311), phosphorylated part of the neurofilament (SMI-312) and the calcium binding proteins Calbindin D28-K, Parvalbumin and Calretinin. Furthermore we checked whether or not a lower temperature of 90 degrees C (instead of boiling the tissue) could be used to achieve the same good staining results. These tests showed that an MW pretreatment in TBS pH 9.0 at full power (boiling) gave the best results. Pretreatment at a lower temperature gave also good results for most (but not all) antibodies when the irradiation time was prolonged to 30 min. The optimal antigen retrieval method also improved the quality of staining with an antibody against neuropeptide Y (NPY), for which, until now, no antigen retrieval appeared to be necessary, even after a long duration of fixation.
Evers
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Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research.
Uylings
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D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis; D001923:Brain Chemistry / Q000528:radiation effects*; D064032:Calbindin 2; D064026:Calbindins; D002648:Child; D002675:Child, Preschool; D005260:Female; D005333:Fetus; D005557:Formaldehyde; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods*; D007223:Infant; D007231:Infant, Newborn; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000276:immunology; D008872:Microwaves; D008875:Middle Aged; D009478:Neuropeptide Y / Q000032:analysis / Q000276:immunology; D010320:Parvalbumins / Q000032:analysis / Q000276:immunology; D064030:S100 Calcium Binding Protein G / Q000032:analysis / Q000276:immunology; D014021:Tissue Preservation
D000941:Antigens; C576764:CALB2 protein, human; D064032:Calbindin 2; D064026:Calbindins; D008869:Microtubule-Associated Proteins; D009478:Neuropeptide Y; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; D005557:Formaldehyde