Electron microscopical demonstration of horseradish peroxidase by use of tetramethylbenzidine as chromogen and sodium tungstate as stabilizer (TMB-ST method): a tracing method with high sensitivity and well preserved ultrastructural tissue.
Until now methods using tetramethylbenzidine (TMB) for electron microscopy (TMB-EM methods) are all unable to provide a maximum demonstration of transported horseradish peroxidase (HRP) while maintaining good ultrastructural tissue preservation. In order to solve this problem, we have attempted to adapt a newly developed, highly sensitive TMB method using sodium tungstate (ST) as the stabilizer (TMB-ST method) for HRP electron microscopic retrograde and anterograde fiber tracing. The present study shows that the TMB-ST method combined with diaminobenzidine-cobalt (DAB-Co) is more sensitive than existing TMB-EM methods and that ultrastructural details are well preserved with this combined method. The resultant reaction product complex after osmication is stable and is observed as characteristic crystal-like structures which are extremely electron dense and often aggregated into clumps. In contrast, the TMB-ST method without the DAB-Co step frequently produces a moderate electron-dense reaction product. Therefore, we recommend the TMB-ST method combined with DAB-Co for HRP electron microscopy.
Gu
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Y
Department of Anatomy, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China.
Chen
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Y
Ye
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D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000818:Animals; D001560:Benzidines / Q000737:chemistry*; D001921:Brain / Q000033:anatomy & histology / Q000648:ultrastructure; D006651:Histocytochemistry / Q000379:methods*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D008854:Microscopy, Electron; D051381:Rats; D017207:Rats, Sprague-Dawley; D014414:Tungsten / Q000737:chemistry*; D017973:Tungsten Compounds
D001560:Benzidines; D017973:Tungsten Compounds; D015100:3,3'-Diaminobenzidine; C021758:3,3',5,5'-tetramethylbenzidine; C025399:sodium tungstate(VI); D006735:Horseradish Peroxidase; D014414:Tungsten