Slowfade antifade solution

To visualize where cultivable S. symbiotica strain is located in the plant, fluorescence in situ hybridization (FISH) was performed as previously described in (Ryuichi Koga, 2009 (link); Caspi-Fluger et al., 2012 (link)). Visualization was performed on plants having undergone the same procedure as the watering experiment and the horizontal transmission test. Six fresh leaves and stems infested with cultivable S. symbiotica for 7 days by the watering experiments and twelve fresh leaves infested with the bacterium for 7 days by infected aphids of the transmission test were thinly sliced vertically with a sterile razor blade to recover part of the midrib. The samples were directly fixed in Carnoy solution at room temperature overnight. After fixation, specimens were bleached in alcoholic 6% H₂O₂ solution for 2 days in complete darkness and then hybridized overnight in hybridization buffer containing the fluorescent probe (10 pmol/ml): Cy3-PASSisR (5′-Cy3-CCCGACTTTATCGCTGGC-3′) targeting 16S rRNA of S. symbiotica. After washing, stained samples were mounted in SlowFade antifade solution (Invitrogen), and observed under a Zeiss LSM 710 confocal microscope. Negatives controls consisted of uninfected leaves and stems and no-probe staining.

Human BM-mesenchymal stem cells (MSCs) (Lonza) were infected with a lentivirus encoding GFP, and Muse cells were collected as SSEA3⁺ cells. For immunofluorescence staining, mouse astrocytes and Muse/non-Muse cells (10,000 cells each) were co-cultured overnight in two-well chamber slides (Nalge Nunc International). The next day, the medium was discarded and several concentrations of Stx2 and 1 μg/mL purified LPS derived from E. coli O55:B5 (Sigma-Aldrich) were used to induce reactive astrocytes. After 6 or 12 h, cells were fixed with 4% PFA for 30 min and then washed twice with PBS. Cells were blocked with blocking buffer (5% normal goat serum/0.3% Triton X-100 in PBS) for 1 h at room temperature and immunostained overnight at 4°C with an anti-GFAP mouse monoclonal antibody (Alexa Fluor 594, #8152; Cell Signaling Technology) diluted by a factor of 100 with dilution buffer (1% BSA/0.3% Triton X-100 in PBS). Samples were stained with DAPI, washed twice with PBS, and mounted with SlowFade antifade solution (Invitrogen). Specimens were examined under the BZ-X700 HS All-in-One Fluorescence Microscope (Keyence, Osaka, Japan).