Anti fgf2

For western blotting, equal amounts of proteins were separated and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then incubated with the following primary antibodies: anti-FGF2 (1:1000; Millipore); anti-p-Akt (1:1000; Thr 473; Cell Signaling Technology, Beverly, MA, USA); anti-Akt (1:1000; Cell Signaling Technology); anti-p-ERK (1:1000; Thr 202/Tyr204; Cell Signaling Technology); and anti-ERK (1:1000; Cell Signaling Technology). An anti-α-tubulin antibody (1:1000; Sigma) was used as a protein loading control. For immunofluorescent staining, cells were grown on coverslips (Thermo Fisher Scientific, Rochester, NY, USA) for 24 h and then incubated with an anti-FGF2 antibody (1:200; Millipore) followed by incubation with an Alexa Fluor 594 IgG antibody (Life Technologies, Carlsbad, CA, USA). The cells were then counterstained with DAPI, and images were captured using a confocal laser scanning microscope (Olympus FV1000, Olympus, Tokyo, Japan).

Animals were perfused, and the eyes with attached nerves were dissected and transferred to sucrose solutions as in the previous item. Tissue was embedded in OCT and sectioned at 20 µm thickness. Tissue sections were rinsed with 0.1% PBST and incubated in 5% normal goat serum (Sigma-Aldrich Co) for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. Sections were then washed in 0.1% PBST and then incubated with secondary antibodies and TO-PRO-3 (1∶1000, Invitrogen Inc.) for 2 h at room temperature. Slides were rinsed in PBS and mounted with VectaShield (Vector Laboratories). Primary antibodies used were anti-dextran (mouse, 1∶500, StemCell Technologies), anti-IBA1 (rabbit, 1∶400, Wako Pure Chemical Industries, Osaka, Japan), anti-IL-1β (rabbit, 1∶100, Peprotech, London, UK) and anti-FGF-2 (mouse, 1∶200, Millipore). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG. Fluorescent samples were analyzed under a confocal microscope (Zeiss LSM 510 Meta). For the Prussian blue reaction and documentation, the procedure was similar to that described for cells on coverslips.