[Optimal fixation for the detection of anti-neuronal antibody by immunohistochemistry in the paraneoplastic syndrome].
Paraneoplastic syndrome(PS) associated anti-neuronal autoantibodies are characterized by their antigen molecular weight determined by Western blot and immunostaining pattern recognized through immunohistochemistry. We investigated immunohistochemical fixatives for their sensitivity in the detection of anti-neuronal nuclear antibody(Hu). Serum used for this study was taken from a patient with anti-Hu antibody seropositivity, which was ascertained by recombinant Hu protein. Western blot analysis produced 37kDa band. We examined six fixative conditions: immersion fixed with acetone, Bouin's solution, Sakura rapid fixative("Ufix'), and perfusion fixed with 2%, 4%, 8% paraformaldehyde (PFA) on the basis of each immunoreactivity in a rat cerebellum, brain stem and liver. The optimal fixation for detecting anti-Hu antibody was perfusion fixed with 2% PFA, that reacted conspicuously with nucleus but not nucleolus of neurons. The perfusion method proved superior to immersion in immunostaining intensity. With immersion fixation, specific immunostaining pattern lessened and differentiation from cytoplasm decreased. With various concentrations of PFA, immuno-reactivity with nucleus at 2% PFA was similar to 4%, although serum optimal dilution at 2% was slightly greater than 4%. The variety of staining patterns of anti-Hu antibody is closely related to the vulnerability of neuronal antigens to the fixatives. The detection of anti-neuronal antibodies is important for early diagnosis and treatment of occult tumors. The immunostaining method is a useful and sensitive way to research these antibodies. Therefore, it is essential to consider the selection of fixation and the preservation of the antigenicity in evaluating immunohistochemical hallmarks.
Kanazawa
N
N
Research Division of East Hospital School of Medicine, Kitasato University, Sagamihara.
Tomiyasu
K
K
Ito
H
H
Irie
S
S
Saito
T
T
Kowa
H
H
D000818:Animals; D000974:Antibodies, Antinuclear / Q000097:blood*; D015153:Blotting, Western; D051959:ELAV Proteins; D006801:Humans; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000276:immunology*; D010257:Paraneoplastic Syndromes / Q000276:immunology*; D016601:RNA-Binding Proteins / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D016707:Tissue Fixation
D000974:Antibodies, Antinuclear; D051959:ELAV Proteins; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins