1 mg/kg three times a week until the day of sacrifice. A third group (n = 10) received intraperitoneal injection of physiological saline three times a week. These doses were selected based upon previous studies that demonstrated the effectiveness of the drug in other disease models [59–61]. Retinal Immunohistochemistry For retinal cryosection immunostaining, sections through the retina (10 mm) with the optic nerve attached were pre-blocked (PBS containing 10% goat serum, 0.5% gelatin, 3% BSA, and 0.2% Tween-20) and then incubated with rabbit anti-Iba-1 antibody (1:500; Wako Chemicals USA Inc., Richmond, VA) as a marker for microglia, anti-neurofilament light chain monoclonal antibody (1:1000; Thermo Scientific), and/or anti-neurofilament middle chain monoclonal antibody (1:1000; Cell Signaling Danvers, MA) overnight at 4uC. Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) or Alexa Fluor 546- conjugated donkey anti rabbit IgG (Invitrogen) were used as secondary antibodies and incubated at room temperature for 1 hour prior to mounting in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). For whole-mount immunostaining, rat eyes were enucleated immediately after transcardiac perfusion. Post-fixed retinas were washed several times in fresh PBS, transferred to multi-well plates, and blocked in 10% normal goat serum (Sigma-Aldrich, St. Louis, MO) with 0.5% Triton X-100 in 0.1 ml PBS overnight. The retinas were then incubated in the same medium containing antibody TUJ1 (1:500; mouse anti-neuronal class III b-Tubulin, Covance, Berkeley, CA), anti-NeuN (1:500; made in mouse, clone A60, Chemicon, Temecula, CA), mouse monoclonal antibody to TNF-a (1:200; Abcam, Cambridge, MA), and/or rabbit anti- Iba-1 antibody (1:500; Wako Chemicals USA Inc., Richmond, VA) for 1 day at 4uC on a shaking platform. After three further washes, retinas were incubated with a goat antibody to mouse IgG conjugated to Alexa Fluor 546 and a donkey antibody to rabbit IgG conjugated to Alexa Fluor 488 diluted in blocking medium (1:400; Invitrogen) overnight at 4uC and flat-mounted onto slides after making four radial slits and cover- slipped in Vectashield mounting medium. Assessment of RGC Survival Surviving RGCs were quantified in retinal whole mount preparations that were double-immunolabeled with the TUJ1 antibody directed against bIII tubulin and anti-NeuN. Within the ganglion cell layer, bIII tubulin is expressed only in RGCs, and thus immunostaining for this antigen can be used to distinguish RGCs from the many amacrine cells that are also in this layer [62,63]. TUJ1- and NeuN-immunofluorescent cells in the ganglion cell layer were visualized with a Zeiss LSM 510 META laser scanning confocal microscope using a 206 objective and were counted manually in eight confocal fields in each quadrant of the retina using ImageJ software and the cell counter plugin. We counted four fields in the center near the ONH and four in the periphery, and then averaged the 32 counts for each retina to 2 obtain RGCs per mm . Assessment of Optic Nerve Axon Survival RGC axons were quantified in the retrobulbar optic nerve 10 mm from the globe under light microscopy. At least three sections were used from each of six rats for each condition. Optic nerves were placed immediately into fixative consisting of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer with 0.08 M CaCl2 overnight at 4uC. The tissue was washed in 0.1 M cacodylate buffer and postfixed in 2% aqueous OsO4. Segments were dehydrated in graded alcohols and embedded in Etanercept Prevents Glaucomatous Neurodegeneration Epon. One-micron sections were cut and stained with 1% toluidine blue in 1% borate buffer. Five regions from each section were chosen for quantitation of axon density. In brief, 1006 photomicrographs of each section were collected using an Olympus Povis AX70 microscope (Olympus Optical, Tokyo, Japan). Axons were counted semi-automatically with a method described previously [64]. The entire surface area of the nerve was represented in each sample and axon density was calculated as the mean across the random micrographs. Measurements of the cross- sectional area of each nerve were combined with axon density to estimate the total number of axons per nerve. Evaluating Microglial Activation Using whole-mounted retinas, we classified the activation of Iba-1-positive microglia as described [25] using four stages. Stage 1 are resting microglia, which have rod-shaped somata with fine, ramified processes; Stage 2 are activated ramified microglia with elongated cell bodies and long, thicker processes; Stage 3 are amoeboid microglia with a round-shaped cell bodies and short, thick, stout processes; Stage 4 are phagocytic cells with round- shaped somata, vacuolated cytoplasm, and lacking any processes observed at the light microscopic level. In all whole-mounts, photos were taken at 86 magnification with the ONH in the center. The numbers of Iba-1 positive cells with the characteristics of stage 3 or 4 activation were counted manually in individual confocal fields using NIH ImageJ software and the cell counter plugin. Colocalization of TNF-a and Microglia Retinal microglia and astrocytes were visualized in retinal whole-mounts by immunostaining with antibodies directed against Iba-1 and GFAP as previously described. The antibody to TNF-a was used for colocalization studies as specified in the manufactur- er’s recommendations. Whole-mounts were visualized using a Leica SP5 confocal microscope with 106 and 636 objectives. The fluorescence of individual fluorochromes was captured separately in sequential mode (diode 405, argon 488, HeNe 543, HeNe 633) after optimization to reduce bleed-through between channels using Leica software. A stack of optical sections was taken at intervals of 0.16 mm. Western Blot Analysis Retinas from experimental eyes with chronic OHT and control eyes were dissected from the RPE-choroid at designated time points after EVC. Samples were separated electrophoret- ically on NuPAGE 4–12% Bis-Tris Gels (Invitrogen) and transferred onto PVDF membrane (0.45 mm; Millipore, Bill- erica, MA). After blocking with 3% nonfat dried milk, membranes were incubated overnight with a primary antibody against TNF-a (1:100, Cell Signaling, Danvers, MA), neurofil- ament light chain (monoclonal antibody, 1:1000; Thermo Scientific), and neurofilament middle chain (monoclonal anti- body, 1:1000; Cell Signaling). The blotted membranes were then incubated for 30 min at room temperature with an HRP- conjugated secondary antibody directed against rabbit IgG (1:10,000; Jackson ImmunoResearch). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) and exposed onto Fuji RX film (Fuji-Film, Tokyo, Japan). Co-immunoprecipitation To investigate whether we could detect Etanercept in the rodent retina, immunoprecipitation (IP) was performed as previously described. [65] Briefly, C57BL/6 mice (n = 2) received a single PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40065