Tsl aobs sp5

After immunomagnetic cell separation, 5 × 10⁴ Glast+ cells from P7 and P28 were placed on PDL- and laminin-pretreated cover slips in a drop (70 μl) of Neurobasal media and kept in a CO₂ incubator for 30 min to allow the cells to attach. After this, the cells were fixed in 4% PFA and blocked with 5% normal donkey serum with 0.15% Tween-20 in PBS at pH 7.4. Cells were then incubated with the anti-Glul antibody and anti-Gfap antibody (Table 1) followed by species-specific fluorescent secondary antibodies (Thermo Fisher Scientific, Carlsbad, CA). Negative controls were incubated with the secondary antibody only. DAPI was used to visualize the nucleus of the cells. Imaging was performed with a confocal laser microscope (Leica TSL AOBS SP5; Leica Microsystems). Individual cover slips were sampled randomly to collect a total of 10 images using a 20X objective lens. The Glul (glutamine synthetase, GS; Müller glia marker), Gfap (astrocyte marker), and DAPI positive cells (total cell number) were counted using ImageJ software. The percentage of Glul and Gfap positive cells, relative to the total number of cells, was calculated.

Cultured primary RGCs were fixed in 4% PF and blocked in buffer containing 5% donkey serum, 0.15% Tween-20 and 1xPBS (pH 7.4). Cells were then incubated with anti-Tubb3 antibody (β-tubulin III antibody, 1:500; Covance, Princeton, NJ), anti-Ripk1 and anti-Ripk3 antibodies (1:250; both from GeneTex, Inc, Irvine, CA), followed by species-specific secondary fluorescent antibodies (Life Technologies, Grand Island, NY). Negative controls were incubated with secondary antibody only. Imaging was performed with a confocal microscope (Leica TSL AOBS SP5; Leica Microsystems, Exton, PA).