Cy3 conjugated donkey anti guinea pig

Hippocampal neurons grown 1 day in vitro (DIV) were treated with 5 µM of PBP10 or 7.5 µM of WRW4 for 3 days. Control samples were treated with vehicle control (water). Cells were fixed with 3.7% paraformaldehyde (Sigma) at the end of 3 days and permeabilised with 0.3% Triton-X (Sigma). The fixed samples were then rinsed with PBS and blocked with 10% normal goat serum (Merck) at room temperature for 30 to 45 min. Cells were incubated with primary antibodies against MAP2 (polyclonal rabbit anti-MAP2, Synaptic Systems, dilution 1:400) and Tau (polyclonal guinea pig anti-Tau, Synaptic Systems, dilution 1:400) at room temperature for 1 h before three consecutive washes with PBS to remove excess antibodies. Following this, cells were incubated with secondary antibodies (Cy2-conjugated donkey anti-rabbit, dilution 1:200; Cy3-conjugated donkey anti-guinea pig, dilution 1:400; both from Jackson ImmunoResearch) for 1 h at room temperature. Cells were further washed with PBS prior to mounting onto glass slides with Fluoro-Gel II with DAPI (Electron Microscopy Sciences).

To estimate the cell density and specificity of GFP labelling in the whole retina, immunohistochemistry was performed on retinal flat-mounts. Five weeks post-intravitreal injection animals were sacrificed with an overdose of sodium pentobarbital by intraperitoneal injection. The cornea and lens were removed and the eye-cups fixed in 4% paraformaldehyde for 2–3 hours. Retinas were washed in 1 × phosphate-buffered saline (PBS) for 10 min and incubated in blocking buffer (10% normal donkey serum, 0.3% Triton X-100) overnight at 4 °C. Retinas were incubated for 6 days at 4 °C in primary antibodies against RNA binding protein with multiple splicing^{43 (link)–45 (link)} (RBPMS, 1:1000 guinea pig anti-RBPMS, gift from Dr. Nicholas Brecha) and choline acetyltransferase (ChAT, 1:100 goat anti-ChAT, Millipore, Billerica, MA, USA) to identify RGCs and cholinergic amacrine cells, respectively. Retinas were then washed in PBS and incubated overnight at 4 °C in Alexa Fluor 488 (1:400 Alexa Fluor^® 488 conjugated rabbit anti-GFP, Molecular Probes, Eugene, OR, USA), Cy3 (1:1000, Cy3 conjugated donkey anti-guinea pig (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and Alexa Fluor^® 633 (1:1000 Alexa Fluor^® 633 conjugated donkey anti-goat, Molecular Probes). Retinas were rinsed in PBS, mounted with anti-fade fluorescent mounting medium and coverslipped.

IF experiments were performed on slices taken from mice sacrificed at the peak of the EAE from at least two different immunization experiments, similarly to [25 ]. Animals were deeply anesthetized and perfused intracardially with ice-cold 4% paraformaldehyde (PFA).
For ex vivo experiments, after incubation with laquinimod or vhl, cerebellar slices (200 μm) were fixed in 4% PFA and equilibrated with 30% sucrose before cutting 30 μm slices. The following primary antibodies were used overnight at 4 °C in Triton X-100 0.25%: rabbit anti-GFAP (1:500; Dako) and guinea-pig anti-GLT-1 (1:5000; Millipore). AlexaFluor-488-conjugated donkey anti-rabbit (1:200; Invitrogen) and Cy3-conjugated donkey anti-guinea pig (1:200; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Nuclei were stained with DAPI. All images were acquired using an LSM7 Zeiss confocal laser-scanner microscope (Zeiss) with a ×20 (zoom ×1 or ×2) objective. All images had a pixel resolution of 1024 × 1024. The confocal pinhole was kept at 1.0, the gain and the offset were lowered to prevent saturation in the brightest signals, and sequential scanning for each channel was performed. Images were exported in Tiff format and adjusted for brightness and contrast as needed using ImageJ software.