Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods.
In this report a comparison is made of three different visualization methods of rat cervical motoneurons retrogradely labelled with cholera toxin B subunit (CTb). CTb conjugates such as CTb-HRP and CTb-FITC or CTb-TRITC, which can be visualized after histochemical detection and by fluorescence microscopy, respectively. The following results were obtained. (1) Immunochemical detection of CTb with peroxidase and DAB-Ni incubation provides the best labelling of the cell bodies and their processes, whereas immunochemical detection with FITC produces less effective labelling of the dendrites. (2) Histochemical visualization of CTb-HRP conjugate gives results similar to those of CTb immunochemistry but produces a much more granular appearance of the label, which may affect the identification of distal dendrites. In addition, direct electron-microscopic analysis of labelled structures can be achieved. (3) CTb-FITC and CTb-TRITC visualization permit double-labelling experiments but the labelled cells exhibit fluorescence only in their somata and proximal dendrites. (4) Factors other than labelling intensity, e.g. double-labelling, preservation of the label, compatibility with other techniques and even economic reasons must be taken into consideration when a selection of visualization methods is to be made.
Dederen
P J
PJ
Faculty of Medical Sciences, University of Nijmegen, Department of Anatomy and Embryology, The Netherlands.
Gribnau
A A
AA
Curfs
M H
MH
D000818:Animals; D002584:Cervix Uteri / Q000294:innervation; D002772:Cholera Toxin; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005552:Forelimb; D006735:Horseradish Peroxidase; D007150:Immunohistochemistry / Q000379:methods*; D009046:Motor Neurons / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines
D005456:Fluorescent Dyes; D012235:Rhodamines; C052635:cholera toxin, B subunit-horseradish peroxidase; C009434:tetramethylrhodamine isothiocyanate; D002772:Cholera Toxin; D006735:Horseradish Peroxidase; D016650:Fluorescein-5-isothiocyanate