Alexa fluor 488 and 594 conjugated donkey secondary antibodies

Deparaffinized liver sections or frozen brain sections were incubated overnight with antibodies against F4/80 (sc-71085, Santa Cruz), Ly6G (ab25377, Abcam), and TREM2 (sc-373828, Santa Cruz) at 4 °C. After washing with 0.1 M PBS, sections were incubated with Alexa Fluor 488- and 594-conjugated donkey secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; 1:20,000, Invitrogen). The fluorescence intensity of the solution was measured using a BX51-DSU microscope (Olympus). For the measurement of the intensity of immunostained protein from liver or brain sections, 12–16 fields (120 × 120 µm² or 20 × 20 µm²) were randomly selected from each section (n = 4 per group) and measured with i-Solution (IMT i-Solution Inc.).

T4 DNA ligase, NdeI, and EcoRI were obtained from New England Biolabs (Ipswich, MA, USA). Alexa Fluor 488- and 594-conjugated donkey secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Streptozotocin (STZ), D-glucose, and some reagents for the western blot analysis such as mouse anti-glial fibrillary acidic protein (GFAP), β-actin, and α-tubulin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Primary antibodies such as GLP-1, Tau, NF-κBp65, insulin receptor (IR)-β, and calbindin were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Rabbit anti-parvalbumin, GLP-1R, hippocalcin, nuclear p84, and mitochondrial VDAC1 were obtained from Abcam (Cambridge, MA, USA). Isopropyl-β-thiogalactopyranoside (IPTG), LB broth, p-Tau, and 4′,6-diamidino-2-phenylindol (DAPI) were obtained from the Thermo Fisher Scientific (Waltham, MA, USA). Total dynamin related protein1 (Drp1) and optic atrophy1 (OPA) were obtained from BD Bioscience (Franklin Lakes, NJ, USA).

Animals were perfused with 4% PFA at 8 weeks after cell transplantation. The spinal cords were dissected out and postfixed at 4 °C overnight, placed in 30% sucrose for 3 days and embedded in OCT compound (Sakura, Torrance, CA, USA). Tissues were sectioned on a cryostat (CM1950, Leica, Wetzlar, Germany) at a thickness of 30 μm. For antigen recovery, the sectioned tissues were incubated in PBS containing 1% SDS (sodium dodecyl sulfate) for 5 min. Sections were incubated with primary antibodies (Supplementary Table 2) at 4 °C overnight and then stained with Alexa Fluor 488- and 594-conjugated donkey secondary antibodies (Invitrogen) for 2.5 h at room temperature. For BDA tract tracing, tissue samples were incubated with Alexa Fluor 488-conjugated streptavidin (Invitrogen). Nuclei were stained with DAPI for 5 min at room temperature. Sections were covered with mounting medium (Fluoromount-G, Invitrogen, 00-4958-02) and glass coverslips. Immunofluorescence was visualized using a fluorescence microscope (IX71) and confocal laser scanning microscope (LSM 700, Zeiss, Jena, Germany).