Imagej software

B. subtilis cultures were grown as liquid cultures in DSM medium, as described above, and cells were harvested 2 h after the onset of stationary phase. For membrane visualization, the fluorescent dye FM 4–64 (Molecular Probes, Eugene, OR, USA) was used at concentrations of 0.2–1 μg·ml⁻¹. Cells were examined under the microscope on 1% agarose covered slides. When it was necessary to increase the cell density, cells were concentrated by centrifugation (3 min at 2500 rpm) and resuspended in a small volume of supernatant prior to examination by microscopy. All images were obtained with an Olympus BX63 microscope equipped with an sCMOS Zyla-4.2P camera (Andor, Oxford Instruments, Belfast, UK). Olympus CellP imaging software and ImageJ software were used for image acquisition and analysis.

Cells were grown on coverslips and fixed with 3.7%(v/v) paraformaldehyde for 20 min at RT, followed by permeabilization with 0.05%(w/v) Triton-X-100 for 5 minutes. Next, cells were blocked with 3%(w/v) BSA in PBS for 2 h. Primary antibodies (1:100) were diluted in 1%BSA in PBS and incubated for overnight at 4 °C. Cells were stained with Alexa-488 phalloidin and/or Alexa-594 DnaSe1 (Invitrogen, USA) for staining F-actin and/or G-actin, respectively, according to the manufacturer’s protocol. Secondary antibodies used (rabbit anti-mouse Alexa-488/-546, Invitrogen), were diluted in 1%BSA and incubated for 2 h at 4 °C. Nucleus was stained with DAPI (1:10,000). Images were acquired in Olympus fluoview confocal microscope/BX61 using 63×/100× objective. Laser intensities and detector gains were maintained at the same level during all imaging sessions. Z-stack images with an average step size of 0.35 μ were captured and 3D-projections of each stack processed for background correction. Image calculations and colocalization analysis have been done using Olympus fluoview and ImageJ software.

Strains containing Gfp-Met3 or Gfp-Cys3 alleles were induced in several nutritional conditions depending on experimental design. Cells were grown overnight in 5 mL YEPD at 30 °C with 150 rpm rotation, washed with PBS three times, then diluted to OD₆₀₀ = 0.6 (5 mL), and incubated in various nutritional conditions at 30 °C for 2 h. A 1 mL aliquot was removed for microscopy analysis. The cells were fixed in 4% formaldehyde (Sigma) (v/v) diluted in 100 mM potassium phosphate and 0.5 mM MgCl₂ for 10 min at 30 °C and washed twice with 1X PBS. Glass slides were prepared with 4 µL of ProLong with NuckBlue antifade (Thermo Scientific) and 6 µL of the processed sample. Cells were viewed by direct fluorescent microscopy using an Olympus BX51M microscope and analysis was performed using Olympus CellSens, PhotoShop CS6, and ImageJ software. Percentages of nuclear Gfp-Cys3 were obtained by counting the cells in which Gfp and DAPI were overlaid (n > 100 cells/replicate). All microscopy experiments were done in triplicates.

Brain tissues were cut into 35 µm thick slices until the entire structure of the hippocampus was detected. The slices were washed three times with 0.1 M phosphate-buffered saline (PBS) and incubated at room temperature for 20 minutes in 5% bovine serum albumin. Primary (overnight, 4°C) and secondary antibody incubations (one hour, room temperature) were carried out. The slices were washed three times with PBS after each incubation. Primary antibody was mouse anti-NR2B 1 : 200 (ab93610, Abcam). Secondary antibody was goat anti-mouse immunoglobulin G (Alexa Fluor 488) 1 : 2000 (ab150157, Abcam). Slices were coverslipped with a water-based mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (KeyGEN BioTECH, Jiangsu, China). Stained sections were then observed by fluorescence microscopy (Olympus) and processed with ImageJ Software.

To detect the ATP levels within the tombusvirus replication compartments in plant cells, in the case of TBSV, PGK-silenced plants or control plants were agroinfiltrated with plasmids pGD-p33-ATeamYEMK, pGD-DI-72, pGD-35S::RFP-SKL, pGD-35S::p19 and pGD-p92. In the case of CIRV, the leaves were agroinfiltrated with pGD-p36-ATeamYEMK, pGD-35S::AtTim21-RFP, pGD-35S::p19, pGD-DI-72 and pGD-p95. The images were taken at 2.5 or 3.5 days post-agroinfiltration and analyzed with the method described previously [31 (link)]. Confocal FRET images were obtained with an Olympus FV1000 microscope (Olympus America). Cells were excited by a 405 nm laser diode, and CFP and Venus were detected at 480–500 nm and 515–615 nm wavelength ranges, respectively. Each YFP/CFP ratio was calculated by dividing pixel-by-pixel a Venus image with a CFP image using Olympus FLUOVIEW software or ImageJ software.
Additional standard experimental procedures are presented in the supporting information S1 Text.

Paraffin-embedded inflated lungs were deparafinized and rehydrated. Afterward, endogenous peroxidase in tissue were quenched with 3% H₂O₂. The tissue sections were then incubated with antibody according to standard protocols. Images were acquired using an Olympus inverted scope (Olympus, Japan) and staining area were semiquantitatively analyzed with ImageJ software.