Jr. Lukas et al., CARBOCYANINE POSTMORTEM NEURONAL TRACING - INFLUENCE OF DIFFERENT PARAMETERS ON TRACING DISTANCE AND COMBINATION WITH IMMUNOCYTOCHEMISTRY, The Journal of histochemistry and cytochemistry, 46(8), 1998, pp. 901-910
Carbocyanines (Dil, DiA, DiO) are able to travel along membranes by di
ffusion and therefore have been used as postmortem neuronal tracers in
aldehyde-fixed tissues. Surprisingly, detailed data on the influence
of different parameters on tracing distances are still missing. This s
tudy was carried out to optimize tracing procedures and to reveal the
validity of the combination of postmortem tracing with immunocytochemi
stry. Carbocyanine crystals were applied to the cervical spinal cord,
sciatic nerves, and brachial plexuses of humans and guinea pigs. Incub
ation in the dark at 37C for 12-15 weeks proved optimal to achieve lon
gest tracing distances (28.9 +/- 2.2 mm) in human and animal tissues.
Longer incubation times and incubation temperatures higher than 37C di
d not result in longer tracing distances. No differences were evident
between adult acid newborn animals and between central and peripheral
nervous system. The diffusion coefficient for Dil was calculated to be
2.5 x 10(-7) cm(2) sec(-1). After application of Dil to nerves of gui
nea pig extraocular muscles, Dil-positive afferent perikarya were obse
rved iri the anteromedial part of the trigeminal ganglion. These perik
arya were identified by calcitonin gene-related peptide immunoreactivi
ty (CG RP-IR). The percentage of CGRP-IR neurons after tracing was con
cordant with the percentage of CGRP-IR in trigeminal ganglia exclusive
ly processed for CGRP-IR without previous postmortem tracing. These re
sults demonstrate carbocyanines to be specific tracers for exact neuro
nal mapping studies.