Goat anti rat igg alexa 488

Mice were anesthetized with Avertin and were perfused with cold PBS and subsequently with 4% PFA in PBS solution. The brains were removed, post-fixed with 4% PFA for 24 h and transferred to 30% sucrose solution. Frozen brains were cut with a thickness of 30 μm in a cryostat. Slices were preserved in 30%PEG 30% sucrose PBS. The brain slices were washed three times with Tris-buffered saline (TBS) and were then blocked with TBS-blocking solution (1% bovine serum albumin, 0.2% skim milk and 0.3% Triton X-100 in TBS) for 1 h and incubated with the following primary antibodies in TBS-blocking solution overnight on a shaker at 4 °C. Rabbit anti-S1P2R antibody (1:100, Proteintech Group Inc., Chicago, IL) and rat anti-CD31 antibody (1:100, BD Biosciences, Pharmingen, San Jose, CA) were used as primary antibodies. Sections were washed three times with TBS and then they were incubated with donkey anti-rabbit IgG Alexa-594 and goat anti-rat IgG Alexa-488 (1:250, Life Technologies, Molecular Probes, Grand Island, NY). Brain slices were stained with DAPI for 7 min and were mounted onto slides. Samples were observed on a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss Inc.). Images were captured using Zen LE software (Carl Zeiss). The specificity of the S1PR2 antibody was confirmed by lack of signal in the S1pr2^−/− brain sections.

Fixed retinal sections were washed with PBS and blocked in 3% bovine serum albumin in PBS containing 0.3% Triton X-100 (PBS-T). The sections were then incubated with anti-glutamine synthetase (rat monoclonal, 1:500, Abcam), anti-GFAP (rabbit polyclonal, 1:500, Dako), and anti-Ki67 (mouse monoclonal, 1:500; BD Biosciences) for 2 h at room temperature. After washing with PBS-T, primary antibodies were visualized using goat anti-rat IgG (Alexa488, 1:1000, Life Technologies Japan). Fluorescence images were captured using a fluorescence microscope (BX53, Olympus) equipped with a cooled CCD camera (DP80, Olympus) or a confocal microscope (LSM880, Zeiss Japan).

The half-brains were fixed overnight, equilibrated in 30% sucrose in PB overnight, frozen in cold isopentane, and stored at −80 °C. The frozen brains were then cryosectioned into 30 μm sections on a −25 °C freezing stage microtome and free-floating sections were stored in a cryoprotectant solution until assayed. For immunofluorescence protocols, sections were washed to remove the cryoprotectant, blocked with 10% normal goat serum, and incubated in primary antibody for 48 h at 4 °C. For Aβ immunostaining, sections were incubated with 70% formic acid for 3 min after the first wash and washed prior to the blocking step. After primary antibody incubation, the sections were washed and incubated with secondary antibodies bound to Alexa fluorophores (Invitrogen) for 2 h at room temperature. Sections were mounted and cover slipped with Prolong Gold (Thermo Fisher, Waltham, MA, USA). Primary and secondary antibody dilutions were as follows: biotinylated 6E10 (BioLegend #803008) 1:2000; rabbit anti-Iba1 (Wako #019-19741) 1:3000; rat anti-LAMP1 (eBioscience #14-1071-82) 1:2000; rat anti-mDectin-1 (CLEC7A, InvivoGen #mabg-mdect, San Diego, CA, USA) 1:800; goat anti-rat IgG Alexa 488 (Invitrogen #A-11006) 1:1000; goat anti-rabbit IgG Alexa 647 (Invitrogen #A-21245) 1:1500, and Streptavidin Alexa 594 or 488 (Invitrogen #S11227) 1:1000.