For immunoperoxidase staining, retinal sections from normal and glaucomatous eyes were deparaffinized, rehydrated, and pretreated with 3% hydrogen peroxide in methanol to decrease endogenous peroxidase activity. After the sections were washed with phosphate-buffered saline solution containing 0.1% bovine serum albumin, they were incubated with 20% inactivated normal donkey serum (Chemicon International, Inc., Temecula, CA) for 30 minutes at room temperature to block background staining. The sections were then incubated with monoclonal antibodies against glial fibrillary acidic protein (GFAP; 1:400, Sigma-Aldrich, St. Louis, MO), and HLA-DR (1:100; Accurate Chemical, Westbury, NY) to identify astrocytes and microglial cells, respectively. A mouse antibody against α-smooth muscle actin (1:800; Sigma-Aldrich) was used to identify pericytes. In addition, we used monoclonal antibodies against phospho-ERK1/ERK2 MAPK (recognizes dual phosphorylated protein at threonine 202 and tyrosine 204, 1:400) and phospho-SAPK/JNK (recognizes dual phosphorylated protein at threonine 183 and tyrosine 185, 1:1000) and a polyclonal antibody against phospho-p38 MAPK (recognizes dual phosphorylated protein at threonine 180 and tyrosine 182, 1:1000). All the phosphorylation site-specific MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). After the sections were washed, they were incubated with the biotinylated secondary antibodies (1:400; Chemicon International, Inc.) for 1 hour at room temperature and then with avidin-biotin complex (ABC solution; Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. After several washes, color was developed by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) used as a cosubstrate for 5 to 7 minutes. Sections were counterstained with hematoxylin and mounted (Permount; Fischer Scientific, Pittsburgh, PA). The primary antibody was eliminated from the incubation medium, or mouse serum (Sigma-Aldrich) was used to replace the primary antibody to serve as negative control. Slides were examined with a microscope (Nikon, Tokyo, Japan), and images were recorded by digital photomicrography (Optronics, Goleta, CA).
For double immunofluorescence labeling, sections were incubated with a mixture of mouse and rabbit antibodies at 1:400 dilution for 1 hour at room temperature. Primary antibodies used in double immunolabeling included the mouse antibodies against MAPKs described earlier and rabbit antibodies against GFAP (1:400), HLA-DR (1:100), α-smooth muscle actin (1:800), or Brn-3a (1:400, Chemicon International, Inc.). Brn-3a is a member of the POU-domain genes, which are known to be expressed by most ganglion cells across a variety of mammalian species.
31 The sections were then incubated with a mixture of rhodamine-red and Oregon-green–labeled secondary antibodies (1:400; Molecular Probes, Eugene, OR) for another 1 hour at room temperature. Negative controls were performed by replacing the primary antibody with 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).