4 6 diamidino 2 phenylindole dapi

Immunofluorescence microscopy was performed on cells grown on coverslips. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100/PBS. After blocking, coverslips were incubated for 1 h at room temperature with the indicated antibodies. For detection of anti-BrdU, after permeabilization with 0.4% Triton X-100/PBS, cells were denatured in 2.5 N HCl for 45 min at room temperature. Alexa Fluor^® 488 conjugated-goat anti mouse and Alexa Fluor^® 594 conjugated-goat anti-rabbit secondary antibodies (Life Technologies) were used at 1:200. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:4000; Serva). Coverslips were observed at 20× objective with the Eclipse 80i Nikon Fluorescence Microscope, equipped with a ViCo system. Images were processed using Photoshop (Adobe) to adjust contrast and brightness. For each time point at least 200 nuclei were examined. Parallel samples incubated with either the appropriate normal serum or only with the secondary antibody confirmed that the observed fluorescence pattern was not attributable to artefacts. Experiments for labelling cellular DNA with EdU or EU were performed by pulse labelling cells with EdU or EU in culture media (10 µM) for 30 min. Detection was performed using Click-iT EdU or EU imaging kits according to the manufacturer's specification (Invitrogen).

In order to identify different retinal cell types, specific immunofluorescence antibodies were applied (n = 5–9 eyes/group; 6 sections/animal, Table 1) [64 (link)]. Briefly, retinal cross sections were blocked with a solution containing 10–20% donkey, 2–3% bovine serum albumin, and/or goat serum and 0.1% Triton-X in PBS. The sections were incubated with primary antibodies at room temperature overnight. Incubation using corresponding secondary antibodies was performed for 1 h on the next day. For the detection of apoptotic cells, a commercially available TUNEL Kit (In Situ Cell Death Detection Kit; Roche; Sigma-Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s instructions. Nuclear staining with 4′,6 diamidino-2-phenylindole (DAPI, Serva Electrophoresis, Heidelberg, Germany) was included to facilitate orientation on the slides. Negative controls were performed for each stain by using secondary antibodies only.

To evaluate the myelin status of the optic nerves 3, 7, and 14 days after immunization, longitudinal sections of the optic nerves were stained against the myelin basic protein (MBP). Briefly, the sections were blocked with 10% goat serum and 0.1% Triton-X in PBS for 60 min. The primary antibody MBP (1:100; Millipore, Darmstadt, Germany) was incubated overnight. The next day, the secondary antibody goat anti-mouse Alexa Fluor 488 (1:500; Invitrogen, Darmstadt, Germany) was added for 60 min. Nuclear staining with 4′,6 diamidino-2-phenylindole (DAPI; Serva Electrophoresis, Heidelberg, Germany) was included. Negative controls were performed by using only the secondary antibody.