Deparaffinized globe sections were stained using the
avidin-biotin-peroxidase complex method as previously
described.
21 Antibodies to NT4/5 (1:250) and BDNF (1:250;
Santa Cruz Biotech, Santa Cruz, CA) were used to detect altered
neurotrophin distribution.
25 26 27 28 Results with these
antibodies were confirmed by immunostaining on other sections from the
same eyes using antibodies to both neurotrophins obtained from the
laboratory of David Kaplan (Montreal Neurologic Institute, Quebec,
Canada).
29 c-
jun antibodies
(c-
jun/AP-1 ab-2, 1:2000, Oncogene, Cambridge, MA and
c-
jun/AP-1 (N)-G , 1:1000; Santa Cruz Biotech) were used to
detect early response gene activation.
11 12 13 14 Antibodies to
phosphorylated neurofilaments (PNF; SMI 34, 1:15,000; Sternberger
Monoclonals, Baltimore, MD) and growth-associated protein-43 (GAP-43,
1:1000; Boehringer–Mannheim, Indianapolis, IN) were used to detect
axonal injury, obstructed axonal transport, and perikaryal protein
accumulations.
15 30 31 32 Optic nerve head glial cell
activation and tissue remodeling were evaluated by immunolabeling for
altered gap junctional communication,
33 34 35 36 37 glial
proliferation,
18 38 39 40 cytoskeletal
organization,
41 42 and extracellular matrix
deposition,
21 43 using antibodies to connexin43 (Cx43,
1:250; Transduction Laboratories, Lexington, KY), proliferating cell
nuclear antigen (PCNA, 1:5000; Santa Cruz Biotech), glial intermediate
filament protein (GFAP 1:2000, Dako, Carpinteria, CA), and collagen VI
(1:250; Southern Biotech, Birmingham, AL), respectively. Although the
first three are typical of glial activation after neural injury, the
last appears specific to glaucomatous nerve heads.
44 For
type VI collagen and c-
jun/AP-1 ab-2 antigen retrieval
before immunostaining, we used predigestion with 0.5 mg/ml trypsin, 15
minutes at 37°C, and with 0.1% pepsin in 0.01 N HCl, 30 minutes at
room temperature, respectively. The use of these antibodies for antigen
immunolocalization has been documented and published previously,
referenced in organ systems including the brain and
eye.
11 12 21 29 30 31 38 40 41 42 43 44 45 46 47 In general, sections from
each of the 22 experimental and control eyes were immunolabeled for
each of the nine proteins. With antibodies such as PCNA and
c-
jun, which do not normally label in the retina, optic
nerve head, or nerve, normal labeling intensity and distribution in the
corneal epithelial layer provided an internal control on each section.
In addition, each immunoassay included sections from experimental and
fellow eyes that were exposed to the complete immunohistochemical
labeling process, but with appropriately diluted, normal sera or
immunoglobulin substituted for the primary antibody.