Bx6 microscope

Anesthetized mice were perfused with 10% formalin in buffered saline for 5 min before dissection of the heart and the entire aorta to the iliac bifurcation. The tissues were stored in 10% buffered formalin solution for 1 week. The aortas were opened longitudinally and stained with Oil Red O for 30 min, whereas the top half of the heart was cryopreserved in 4% paraformaldehyde at 4 °C overnight before imbedding in OCT compound for frozen section preparation. Eight cryosections of 7-μm thickness through the aortic valve region were stained with Oil Red O for 15 min and counterstained with hematoxylin for 3 min. Composition of the atherosclerotic lesions was determined by staining serial sections for 1 h with Sirius Red to identify fibrotic areas and immunohistochemical analysis with antibodies against CD68, IL-1β, or nitrotyrosine. Immunohistochemical analysis was performed using VECTASTAIN ABC-HRP kits (Vector Laboratories) according to manufacturer's instructions. Images were obtained using an Olympus BX6 microscope and digitalized for quantitative analysis using ImageJ software as described (88 (link)).

The 11399-WT and 11399-mqsR cells were grown for 7 days on PW broth as described previously and had their OD₆₀₀ standardized to 0.1, and 300 μL aliquots of each sample were inoculated into 2.7 mL of fresh PW broth in glass bottom microwell (MatTek) petri dishes. In total, 8 plates were inoculated for each strain and were grown at 28°C for 15 days. The cells were exposed to 0, 1, 3, or 7 mM of copper (CuSO₄) for 24 h, the supernatant was then discarded, and the adhered cells were gently washed once with 1 mL of PBS buffer. The adhered cells were stained using the LIVE/DEAD BacLight Bacterial Viability kit (Invitrogen) (240 μL of SYTO-9 5 μM + 6 μL of propidium iodide 20 μM) and visualized by fluorescence microscopy using a BX6 microscope (Olympus). The cell lengths were calculated using ImageJ software (http://rsbweb.nih.gov/ij/) to determine the proportion of elongated cells among the longest cells of 11399-mqsR and 11399-WT (>2.0 μm). Only cells longer than 4.0 μm were considered elongated because the length of normal X. fastidiosa cells ranges from 0.9 to 4.0 μm (Almeida et al., 2014 ).

The 11399-WT and 11399-mqsR cells were collected from PWG plates and suspended in 2 mL of PBS buffer to an OD₆₀₀ of 0.6, which was considered time 0. Then, without disturbing the cell suspension, an aliquot of 100 μL of the supernatant of each suspension was taken every hour for 6 h to measure the OD₆₀₀ of the samples. The OD₆₀₀ of the supernatant is lower with increased aggregation because cell aggregates sediment faster at the bottom of the tube.
Additionally, microscopy analysis of the aggregates was carried to 11399-WT and 11399-mqsR cells. In brief, cells were collected as above and after 1 h, without disturbing the cell suspension, aliquots of 100 μL of the supernatant and the sedimented cells of each suspension were taken for analysis. Each aliquot was stained with 100 μL of SYTO-9 5 μM (Invitrogen) for 15 min in the dark. The cells were visualized by fluorescence microscopy using a BX6 microscope (Olympus).