Seven-micrometer longitudinal paraffin sections of rat and monkey
optic nerve heads were collected onto Superfrost Plus slides (Fisher
Scientific; Pittsburgh, PA) before immunolabeling by the
streptavidin-biotin peroxidase technique of Lutty et al.
40 The affinity-purified rabbit polyclonal antibodies were directed
against BDNF and against the neurotrophin receptors TrkB, TrkA, and
TrkC (Santa Cruz Biotechnology, Santa Cruz, CA). For the colabeling of
TrkB and GFAP, an affinity-purified goat polyclonal antibody directed
against TrkB was used (Santa Cruz Biotechnology). The antibody
recognizing human BDNF was raised against a peptide corresponding to
amino acids 128 to 147. Both the rabbit and goat polyclonal TrkB
antibodies were directed against an amino acid sequence 794 to 808,
located within the intracellular portion of the full-length murine
gp145
TrkB receptor molecule. The TrkA antibody was raised
against a peptide that corresponds to amino acids 763 to 777, adjacent
to the carboxyl-terminus of the precursor form of the porcine
gp140
Trk receptor molecule, and the TrkC antibody was
directed against a peptide corresponding to amino acids 798 to 812 of
the precursor form of porcine TrkC, gp140
TrkC. TrkB, TrkA,
and TrkC antibodies were noncrossreactive with one another, as
determined by the supplier with immunoprecipitation. Western blot
analysis confirmed the presence of bands at the appropriate molecular
weight (data not shown). Primary antibodies were applied at 0.5 to 1.0μ
g/ml. Negative controls included nonimmune serum of the same species
as the primary antibody at the same protein concentration, primary
antibody preincubated with fivefold excess control peptide, and
incubation buffer alone. Labeled sections were mounted in glycerol
jelly and viewed by Nomarski optics (Zeiss Axioskop; Carl Zeiss Inc.,
Thornwood, NY).
In addition, a series of rat and monkey optic nerve head sections were
colabeled with goat anti-TrkB and a rabbit polyclonal antibody directed
against bovine GFAP (Dako Corporation, Carpinteria, CA) to ascertain if
TrkB label was localized in astrocytes. For this experiment, goat
anti-TrkB was followed by donkey anti-goat biotinylated secondary
antibody (Santa Cruz Biotechnology) and Cy3-labeled streptavidin
(Jackson ImmunoResearch Laboratories, West Grove, PA), respectively.
After reblocking in normal serum, rabbit anti-GFAP was applied followed
by fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit secondary
antibody (Santa Cruz Biotechnology). Sections were mounted in
Vectashield mounting media (Vector Laboratories, Burlingame, CA) and
viewed on a confocal microscope (Zeiss LSM 410; Carl Zeiss Inc.).
Images of Cy3-TrkB label were collected using a helium-neon laser at
543-nm excitation, with a 570-nm longpass filter, whereas images of
FITC-GFAP were collected using a krypton-argon laser at excitation 488
nm, with a band pass of 505 to 580 nm. Cy3-TrkB images were
pseudocolored red and FITC-GFAP green before their superimposition.
Because retinas from the monkey nerve heads had been removed for other
studies, additional retinal samples from archival control monkey eyes
were labeled with goat anti-trkB as described above. Epifluorescent
images of the Cy3-TrkB label were collected with the Zeiss Axioskop
using a rhodamine filter (Carl Zeiss Inc.).