Bx51wi

Recordings were done using a T.I.L.L. Photonics imaging system (Martinsried, Germany) coupled to an epifluorescent microscope (Olympus BX-51WI, Olympus, Hamburg, Germany) equipped with a 10x water immersion objective. Images were taken using a 1004 × 1002 pixel 14-bit monochrome CCD camera (Andor iXON) cooled to −70°C. Each measurement consisted of 80 frames at a rate of 5 frames/s (integration time for each frame: 10–15 ms). The excitation light was applied using a monochromator (T.I.L.L. Polychrom V). The microscope was equipped with a GFP-BP filter set composed of a 490 nm dichroic beamsplitter and a 525/550 nm emission filter.

Dhb sp. UNSWDHB cells, previously isolated from a CF‐respiring enrichment culture (Lee et al., 2012), were grown at 30°C anaerobically in 2‐litre flasks containing defined mineral medium with hydrogen as the electron donor (Lee et al., 2012; Wong et al., 2016) and CF as the electron acceptor. The growth of the cells was monitored using cell counting under fluorescence microscopy. Glutaraldehyde and SYBR Green fluorescent stain (Thermo Fisher, USA) were used as the fixing and staining solution respectively. The stained cell suspensions were fixed onto a 1.5% agarose‐coated microscope slide and were imaged on a fluorescent microscope OLYMPUS BX51WI (Olympus‐Lifescience, USA). The medium and all buffers used were prepared using anaerobic techniques and contained 1 mM dithiothreitol (DTT). Working inside an anaerobic chamber Whitley DG250 (Don Whitley, West Yorkshire, UK) under a gas mixture containing 80% N₂, 10% H₂ and 10% CO₂, 2 l of anaerobic cultures were transferred to airtight centrifuge tubes modified with PVC thread tape and harvested by centrifugation at 10 000 g for 45 min at 4°C. The cell pellets were washed in 40 ml of anaerobic 100 mM Tris buffer, pH 7.5, with 5% (w/w) glycerol, 1 mM DTT and 150 mM NaCl and subjected to another round of centrifugation under the same condition. The cell pellets were stored anoxically at −80°C until further use.

Invasion assays were performed using modified Boyden chambers [33 (link)] coated with Matrigel. 8 μM pore transwells (Falcon HTS FluoroBlock Inserts, BD Biosciences, Bedford, MA, cat. # 351152) were pre-coated with 30 μL of BD Matrigel^TM Matrix (BD Biosciences, Bedford, MA, cat. # 354263) for 2 hours at 37°C. Glioma cells (50,000 cells) were placed on top of the membrane in the upper chamber in 150 μL of DMEM and microglia (50,000 cells) were placed in the lower chamber in 500 μL of DMEM. The control group did not contain microglia. Invasion was allowed to proceed for 18 hours at 37°C, 5% CO₂. In migration assays, Matrigel that mimics the extracellular matrix was not used. Migration assays were performed for 5 hours at 37°C, 5% CO₂.
In both assays, migratory cells were fixed with 100% methanol and stained with 40 μg/μL Propidium iodide (PI). Images of the cells that migrated to the lower chamber were captured using an inverted microscope Olympus BX51WI (Olympus, Shinjuku, Tokyo, Japan), Qcolor 3 camera (Olympus) and Q-capture Pro software (Olympus) and the number of migrating and invading cells on the underside of the filter were counted. The mean of the total invading or migrating cells was determined from 3 independent experiments.

The stem height, root length, and area of the fifth leaf [39 ] were measured in 40-day-old plants. The fresh weight of the whole plant was determined with the gravimetric method; the dry weights of the root, stem and fifth leaf were determined after fixation at 110 °C and drying at 70 °C to constant weight. The number of biological replicates was at least 30 plants in each variant.
Fragments of the main root (mature zone) and stem (the fourth internode from the cotyledon leaves) at the age of 40 days were fixed in the mixture of 96% ethanol:glacial acetic acid (3:1, v/v) at 4 °C [40 ]. After 48 h, the samples were washed and stored in 96% ethanol at 4 °C and used for the anatomical analysis. Cross sections (100 µm-thick) were obtained with the freezing microtome MZP-01 (TECHNOM, Ekaterinburg, Russia). Lignin was stained with phloroglucinol–HCl [41 (link)]. The transverse sections of roots and stems were examined under a wide-field microscope Olympus BX51 WI (Olympus Corporation, Tokyo, Japan). Cell and tissue characteristics were studied using SIMAGIS^® Meso-Plant™ software version 2.1 for Windows XP. Five sections of each organ from one plant were analyzed. The total number of studied sections was at least 30 in each variant.

Behavior naïve LV-GFP (n = 3), LV-Cav1 (n = 5), and LV-shCav1 (n = 4) and methamphetamine self-administered LV-GFP (n = 4), LV-Cav1 (n = 4), and LV-shCav1 (n = 3) and virus and behavior naïve (n = 8) rats were anesthetized with isoflurane and killed by rapid decapitation. Brains were quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCI, 26 NaHCO₃, 4 KCI, 1.25 NaHPO₄, 2 CaCl₂, 1 MgCl₂, and 10 glucose bubbled with 95% oxygen and 5% CO₂ [15 (link)]. Brains were trimmed on the dorsal side at an angle of approximately 140° from the horizontal plane and glued to a vibratome base (Leica VT1000S). Thick slices (440 μm) containing the dorsal striatum were obtained and used for recordings. Three-four slices were transferred to a submerged chamber and incubated with oxygenated ACSF at 25 °C for at least 1–1.5 h before initiating recordings. Recordings were made in the dorsal striatum (Figure 4b,c), in a submersion-type recording chamber superfused with oxygenated ACSF at a rate of 2–3 mL/min at 25 °C and positioned on the stage of an upright motorized microscope (Olympus BX51 WI, Scientifica, Clarksburg, NJ, USA) equipped with a back Illuminated sCMOS camera (Prime 95B, Photometrics, Tucson, AZ, USA) and a broad-spectrum LED illuminator (pE-300, CoolLED).