Mab1585

Immunodetection in flat mounts and brain coronal sections was carried out as reported (Galindo-Romero et al., 2011 (link); Nadal-Nicolás et al., 2015 (link)). Primary antibodies were: mouse anti-Brn3a (1:500; MAB1585, Merck Millipore; Madrid, Spain), mouse anti-human mitochondria (1:800, ab3298 Abcam, Cambridge, United Kingdom), and rabbit anti-melanopsin (1:1,000; AB-N39 Advanced Targeting Systems ATS, Joure, Netherlands). Secondary detection was carried out with Alexa Fluor-coupled secondary antibodies (1:500; Molecular Probes; Thermo Fisher Scientific, Madrid, Spain). Retinal whole-mounts and brain coronal sections were mounted with anti-fading mounting media.

Euthanized animals underwent transcardial perfusion with a 0.9% saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffer solution. Flat-mounted retinas were prepared according to established procedures (49 (link)). Immunodetection procedures followed previous protocols (50 (link)). Flat-mounted retinas underwent triple immunodetection with mouse anti-Brn3a (1:500; MAB1585, Merck Millipore; Madrid, Spain) and rabbit anti-melanopsin (1:750; UF008 (AB-N39), Advanced Targeting Systems, Carlsbad, CA, USA) antibodies to quantify the total number of vision-forming and non-vision-forming retinal ganglion cells (RGCs), respectively. Additionally, guinea pig anti-Iba1 antibody was used to identify microglial cells or infiltrated macrophages (1:500; 234308, Synaptic Systems, Göttingen, Germany).
Secondary detection employed Alexa Fluor-labeled secondary antibodies (1:500; Molecular Probes; Thermo Fisher Scientific, Madrid, Spain). Retinal whole-mounts were mounted using anti-fading mounting media (H-1200, Vectashield^®, Vector Laboratories Inc., Burlingame, CA, USA).

Animals were perfused transcardially with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Retinas were dissected as flattened whole-mounts as previously described [6 (link)].
Immunodetection was carried out as previously reported [4 (link)]. RGCs were detected with mouse α-Brn3a (brain-specific homeobox/POU domain protein 3A; 1:500; MAB1585, Merck Millipore; Madrid, Spain) and microglial cells, with rabbit α-Iba1 (ionized calcium-binding adapter molecule 1; 1:500; ab178846, Abcam, Cambridge, United Kingdom). Secondary detection was carried out with donkey α-mouse IgG1-Alexa fluor 594 and donkey α-rabbit Alexa 488 (1:500; Molecular Probes; Thermo Fisher Scientific, Madrid, Spain).

After the enucleation of three rats from each treatment group, the retina was dissected with a flattened whole mount. After taking out of the cornea by cutting a circular path along the ora serrata with small scissors, the lens was removed using forceps. Separation of the retina from the eyecup was performed by placing forceps between the retina and the eyecup. After obtaining the entire retina, it was cut into quarters using scissors to incise from the retinal to the optic nerve, as described in previous study [1 (link)]. The retinas were fixed in 4% paraformaldehyde and mounted on a glass coverslip for at least 1 h at room temperature. After washing with PBS, they were incubated in PBS with 1% Triton X-100 at room temperature for 30 min. The retina was blocked in 20% fetal calf serum for 1 h, and incubated with anti-Tuj1 (ab18207; Abcam) or anti-Brn-3a (MAB1585; Millipore) at a 1:10 dilution for overnight at 4 °C. On the next day, the retina was washed with PBS-T and incubated with goat anti-rabbit IgG-fluorescein isothiocyanate and Alexa Fluor 633 antibodies in PBS-T at 1:200 for 2 h. Before mounting on the coverslip, retina was washed again. Images captured using a confocal microscope (LSM 880; Carl Zeiss, Jena, Germany) were used to quantify fluorescence. Two areas were calculated on each retina, and the average values were compared for statistical analysis.