The percentage of infiltrated MAC-1+ cells decreased drastically 4?dpi (Fig. 95%, EtOH 90%, EtOH 80%, EtOH 70%, EtOH 50% and H2O (3 washes, 5?min per wash). Slices were placed in a plastic rack with an antigen retrieval buffer (0.1?M NaCitrate and 0.3%Triton X-100 in PBS) and boiled for 4 in a domestic microwave. After a wash with cold water, sections were blocked in 10% normal goat serum (NGS) for 30?min and in 1% NGS for an additional 30?min. Retinal sections were incubated overnight at 4?C with primary antibodies diluted in PBS. They were then washed with PBS and incubated with secondary antibodies for 1?h at room temperature. For retinal flat mount immunostaining, whole retinae were dissected and fixed for 1?h with 4% PFA. They were then permeabilized and blocked (10% NGS, 0.3% Triton X-100 in PBS), prior to incubation with primary antibodies (two consecutive overnights at 4?C). Retinae were then washed with PBS and incubated with secondary antibodies. TUNEL staining was performed according to manufacturer’s instructions (In Situ Cell Death Detection Kit, Fluorescein). Briefly, retinal flat mounts were permeabilized and blocked (10% NGS, 0.3% Triton X-100 in PBS). They were then incubated with the TUNEL reaction mixture at 37?C. DAPI was also used to stain for cell nuclei. For immuno-TUNEL staining, we first performed immunostaining with primary antibodies, as described above. We then proceeded with the TUNEL reaction, and, lastly, with the secondary antibody staining. The list of primary antibodies used for both retinal flat mounts and sections can be found in Table S2. We used the following secondary antibodies: anti-chicken Alexa Fluor 488, anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 568, anti-mouse Alexa Fluor 647 and anti-rabbit Alexa Fluor 633. All secondary antibodies were provided by Molecular Probes (Invitrogen) and used 1:1000 in PBS. DAPI was also used to stain for cell nuclei. Both retinal flat mounts and sections were mounted with Vectashield (Vector Laboratories, 42 Burlingame, CA, USA) and imaged using either Leica laser SP5 or S130 SP8 confocal microscopy systems. 2.8. Image Processing and Quantification Images from both sections and whole retinal flat mounts were processed with the ImageJ software (US National Institutes of Health, Bethesda, Md., USA). Quantifications were based on analysis of at least three animals. We analyzed a minimum of two sections per mouse, and three random fields per section. For S130 each flat mount, we imaged at least three random fields. To quantify the number of YFP+ cells differentiating into ganglion-amacrine neurons in flat mounts, YFP+ total cells and double positive YFP+/CALR+ cells were counted in at least five random fields per animal (20 objective). The transdifferentiation rate was expressed as the percentage of YFP+/CALR+ cells over the total YFP+ cells present in each field. Similarly, the number of proliferating MGCs (Fig. 1d, S1d) was represented as the percentage of phH3+/YFP+ or PCNA+/YFP+ cells over the total YFP+ cells counted in each imaged field. Open in a separate window Fig. 1 Mller glial cells (MGCs) undergo reprogramming and differentiate into CALR+ cells following NMDA-damage. (a) Experimental scheme. We used transgenic GFAP-Cre/R26Y mice. In these mice, ubiquitous expression of YFP is impeded by the presence of a floxed-STOP codon, which can be excised by Cre recombinase. Expression of Cre recombinase is driven by the glial-specific GFAP promoter. As a consequence, the YFP reporter allows to trace glial cells. We injected NMDA in the right eyes to induce retinal degeneration. Left eyes were injected with PBS, as controls. We characterized YFP+ cells at various time-points post-injection. (b) Representative immunostaining of retinal sections harvested from GFAP-Cre/R26Y mice sacrificed 24?hpi and 4?dpi. Higher magnification images (from the areas enclosed by the white boxes) are shown in the bottom panel. YFP+ cells (green) are also positive for GS (red), a well-known glial marker (onl, outer nuclear layer; inl, inner nuclear layer; gc, ganglion cells layer). Scale bar: 100?m. (c) RT-PCR expression analysis of neural stem cell and retinal progenitor genes using total RNA harvested from FACS-sorted YFP+ cells of either PBS-treated (CTR) or NMDA-damaged (NMDA) retinae of GFAP-Cre/R26Y mice, 24?hpi and 4?dpi. Transcript levels are expressed as fold-changes relative to YFP+ cells sorted from PBS-injected retinae. Data are represented as mean??S.E.M. (n?=?4). S130 Statistical analysis is based on unpaired Student’s and (Fig. 1c, left panel). Some of these trends were maintained 4?dpi (e.g. expression was strongly upregulated 24?hpi, and its levels Rabbit polyclonal to AGO2 remained high 4?dpi (Fig. S1d). Since Cyclin D1 is expressed.