Blot analyses and immunocytochemistry of neural antigens with digoxigenylated primary and secondary antibodies.
While the digoxigenin-anti-digoxigenin (DIG) method is currently the preferred tool for non-radioactive in situ hybridization this study extends its application field to Western blotting of proteins and summarizes advantageous properties of digoxigenylated antibodies in immunocytochemistry. An established protocol for the preparation of digoxigenylated primary antibodies is complemented by dot blot analyses confirming the high sensitivity of hapten-anti-hapten techniques based on primary digoxigenylated antibodies. The comparative Western blot analysis of calcium-binding proteins in nervous tissue is used as an example to show the highly specific detection of relevant antigens with unmodified primary antibodies, digoxigenylated secondary antibodies and anti-digoxigenin-peroxidase conjugates. The application of the DIG technology seems to be especially indicated in tissues containing high amounts of endogenous biotin-bearing proteins which might induce false-positive staining in conventional streptavidin/biotin techniques. Finally, the previously shown suitability of digoxigenylated antibodies for different immunocytochemical procedures is completed here by examples for sensitive single immunoperoxidase staining of neural markers in rat brain and for carbocyanine double immunofluorescence labelling of senile plaques in old rhesus monkeys.
Härtig
W
W
Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Germany.
Kirazov
L
L
Brückner
G
G
Holzer
M
M
Gärtner
U
U
Bigl
V
V
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001253:Astrocytes / Q000166:cytology*; D001710:Biotin; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology*; D064032:Calbindin 2; D064026:Calbindins; D004076:Digoxigenin; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis*; D007074:Immunoglobulin G / Q000302:isolation & purification; D007150:Immunohistochemistry / Q000379:methods; D007202:Indicators and Reagents; D008253:Macaca mulatta; D008297:Male; D009419:Nerve Tissue Proteins / Q000032:analysis*; D010296:Parietal Lobe / Q000166:cytology; D058225:Plaque, Amyloid / Q000648:ultrastructure*; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000032:analysis; D019809:Streptavidin
D000906:Antibodies; D064032:Calbindin 2; D064026:Calbindins; D005904:Glial Fibrillary Acidic Protein; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D001710:Biotin; D019809:Streptavidin; D004076:Digoxigenin