For immunoperoxidase staining, sections from normal and
glaucomatous eyes were deparaffinized, rehydrated, and pretreated with
0.3% hydrogen peroxide in phosphate-buffered saline solution to
decrease endogenous peroxidase activity. Monoclonal antibodies against
TNF-α or TNF-α receptor-1 (2 μg/ml; R&D Systems, Minneapolis, MN)
were localized by immunoperoxidase, with reagents purchased from Vector
Laboratories (Burlingame, CA). The biotinylated secondary antibody was
incubated with the sections for 30 minutes, washed with
phosphate-buffered saline solution containing 0.1% bovine serum
albumin, and reacted with streptavidin-horseradish peroxidase
conjugated for 30 minutes. After several washes, color was developed by
incubation with 3,3′-diaminobenzidine tetrahydrochloride (Sigma, St.
Louis, MO) as a cosubstrate, for 5 to 7 minutes. Sections were
counterstained with hematoxylin and mounted (Permount; Fischer,
Pittsburgh, PA). For a negative control, nonimmune rabbit and mouse
sera (Sigma) were used to replace the primary antibodies. Slides were
examined in a microscope (Nikon, Tokyo, Japan), and images were
recorded by digital photomicrography (Optronics, Goleta, CA).
To study localization of TNF-α or TNF-α receptor-1 in the retina,
we performed a double-immunofluorescence procedure using antibodies
against specific cell markers. We used monoclonal antibody against
glial fibrillary acidic protein as a marker of glial cells. To identify
retinal ganglion cells, we used a monoclonal antibody to Brn-3a
(Chemicon International, Inc., Temecula, CA) that is a member of the
POU-domain genes and is known to be expressed by most ganglion cells
across a variety of mammalian species.
17 18
For double-immunofluorescence labeling, sections were incubated with a
mixture of mouse and rabbit antibodies at 1:100 dilution for 30
minutes. The sections were then incubated with a mixture of
rhodamine-red and Oregon-green–labeled secondary antibodies
(Molecular Probes, Eugene, OR) for another 30 minutes. Negative
controls were performed by replacing the primary antibody with
nonimmune serum or by incubating sections with each primary antibody
followed by the inappropriate secondary antibody, to determine that
each secondary antibody was specific to the species it was raised
against. Slides were examined in a fluorescence microscope (Nikon) and
images were recorded by digital photomicrography (Optronics).