Tissue fixation methods alter the immunohistochemical demonstrability of neurofilament proteins, synaptophysin, and glial fibrillary acidic protein in human cerebellum.
This study has examined the effect of postmortem autolysis, type, and duration of fixation on neurofilament, synaptophysin, and glial fibrillary acidic protein (GFAP) antigen decay as demonstrated by immunohistochemistry, using a streptavidin-biotin peroxidase method. The system used consisted of 5 normal cerebellar cortices. Time intervals, temperature, mode of fixation and storage, and staining technique were well controlled. Anti-neurofilament antibodies comprised SMI-31, MNF, and BF-10 against phosphorylated epitopes, and SMI-32 against a non-phosphorylated epitope. Bouin's and B5 fixative, and Sensofix gave best results, whereas formaldehyde and paraformaldehyde fixation gave much lower immunoreactivity. Phosphorylated neurofilament epitopes were less affected by aldehydes than unphosphorylated epitopes. GFAP staining was most consistent after Bouin fixation while the monoclonal antibody was much more sensitive to the fixative used than the polyclonal one. Aspecific background staining increased considerably after a postmortem interval of 24 hours. Synaptophysin immunoreactivity, as demonstrated by SY-38, proved very sensitive to prolonged fixation and was of poor quality following formaldehyde and paraformaldehyde fixation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of autolyzed tissues that have been fixed and stored in different modes and for variable time interval.
Sillevis Smitt
P A
PA
Department of Neurology, Academic Medical Center H-2-214, Amsterdam, The Netherlands.
van der Loos
C
C
Vianney de Jong
J M
JM
Troost
D
D
D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D002525:Cerebellar Cortex / Q000033:anatomy & histology / Q000378:metabolism; D002531:Cerebellum / Q000033:anatomy & histology / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D016900:Neurofilament Proteins / Q000276:immunology / Q000378:metabolism*; D016708:Synaptophysin / Q000276:immunology / Q000378:metabolism*; D016707:Tissue Fixation
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D005904:Glial Fibrillary Acidic Protein; D016900:Neurofilament Proteins; D016708:Synaptophysin