Tsl aobs sp5 confocal microscope

After treatment, the apoptotic and necrotic neurons were labeled using Vybrant Apoptosis Assay Kit (Life Technologies, Grand Island, NY). Cells were imaged using a Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA). Individual glasses were sampled randomly to collect a total of 6 images using a 20X objective lens. The apoptotic and necrotic RGCs were counted semiautomatically using ImageJ software. The percentage of apoptotic and necrotic RGCs relative to the total number of counted cells on the glass was determined. The experiment was repeated at least three times.

After the animals were euthanized, their eyes were enucleated and fixed in 4% PFA. Retinas were removed after one hour and cryoprotected overnight in 30% sucrose. The following day, the retinas were freeze-thawed three times, rinsed for 3 × 10 minutes in 0.1 M Tris buffer, blocked by 5% donkey serum and 0.1% Triton X-100 in 0.1 M Tris buffer for one hour, and then incubated overnight with either beta III Tubulin antibody (Tubb3, 1:250, Covance, Denver, PA) or with both Tubb3 and p65 (1:400; sc-372, Santa Cruz Biotechnology, Dallas, TX). The next day, the retinas were rinsed in 0.1 M Tris buffer (3 × 10 minutes), flat-mounted, cover-slipped, and imaged using a Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA). To count the number of surviving RGCs, individual retinas were sampled randomly to collect a total of 20 images from four retinal quadrants using a 20X objective lens. Five images were collected from each quadrant: one from the center, two from the middle, and two from the peripheral regions. Tubb3-positive neurons (RGCs) were counted using ImageJ software. RGC survival was calculated as a percentage of the mean cell density in fellow control eyes.

Fixed retinas were sectioned to a thickness of 100 μm with a vibratome (Leica Microsystems, Exton, PA) and immunostained as described earlier (Dvoriantchikova et al., 2009a ; Dvoriantchikova et al., 2009b (link); Dvoriantchikova et al., 2010b (link)). Briefly, sections were permeabilized with 0.3% Triton X-100 in PBS for one hour, rinsed three times in PBS, blocked in buffer (5% donkey serum, 2% BSA and 0.15% Tween-20 in PBS) for 1 hour and incubated overnight with anti-Ripk1 (1:250) and anti-Ripk3 (1:250) specific antibodies (both from GeneTex, Inc, Irvine, CA) as well as anti-Tubb3 antibody (β-tubulin III antibody, 1:500; Covance, Princeton, NJ), followed by species-specific secondary fluorescent antibodies (Life Technologies, Grand Island, NY). Control sections were incubated without primary antibodies. Imaging was performed with a Leica TSL AOBS SP5 confocal microscope (Leica Microsystems, Exton, PA).