In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies.
Cholinergic basal forebrain neurons (CBFN) expressing the low-affinity neurotrophin receptor p75 (p75(NTR)) were previously selectively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75(NTR), but the applied 192IgG-conjugates recognized p75(NTR) only in rat. The antibody ME 20.4 raised against human p75(NTR) had been shown to cross-react with the receptor in monkey, raccoon, sheep, cat, dog, pig and rabbit. Hence, for in vivo labeling of rabbit CBFN in the present study, ME 20.4 was fluorochromated with Cy3-N-hydroxysuccinimide ester and purified Cy3-ME 20.4 was injected intracerebroventricularly. Two days post-injection, clusters of Cy3-ME 20.4 were found in CBFN displaying choline acetyltrans-ferase-immunoreactivity. Following photoconversion, electron microscopy revealed fluorochromated antibodies in secondary lysosomes. In conclusion, Cy3-ME 20.4 might become an appropriate marker for CBFN in live and fixed tissues of various mammalian species.
Härtig
Wolfgang
W
Paul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany.
Varga
Csaba
C
Kacza
Johannes
J
Grosche
Jens
J
Seeger
Johannes
J
Luiten
Paul G M
PG
Brauer
Kurt
K
Harkany
Tibor
T
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000911:Antibodies, Monoclonal; D002232:Carbocyanines / Q000032:analysis*; D002799:Cholinergic Fibers / Q000737:chemistry* / Q000648:ultrastructure; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis; D000922:Immunotoxins / Q000032:analysis*; D007276:Injections, Intraventricular; D008297:Male; D018613:Microscopy, Confocal; D009699:N-Glycosyl Hydrolases; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D016548:Prosencephalon / Q000737:chemistry* / Q000648:ultrastructure; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D005456:Fluorescent Dyes; D000922:Immunotoxins; C108575:ME20.4 IgG-saporin; D054789:Ribosome Inactivating Proteins, Type 1; C089187:cyanine dye 3; D009699:N-Glycosyl Hydrolases; D000076985:Saporins