Axio imager a2 microscope

The microscopic analysis to determine bacterial cell sizes was performed essentially as previously described (Zeden et al., 2018). Briefly, S. aureus strains LAC*, LAC*dacA::kan, LAC*dacA_G206S, LAC*dacA/opuD (ANG3835) and LAC*dacA/alsT (ANG3838) were grown overnight at 37°C in TSB or TSB supplemented with 0.4 M NaCl where stated. Next day, the cultures were diluted to an OD₆₀₀ of 0.01 and grown for 3 hr at 37°C to mid‐exponential phase (OD₆₀₀ of 0.5–0.9). One hundred microlitres of these cultures were then stained for 20 min at 37°C with Vancomycin‐BODIPY FL used at a final concentration of 2 µg/ml. One and a half microlitres of each sample were spotted onto a thin 1.5% agarose gel patch prepared in H₂O or in 0.4 M NaCl and the bacteria subsequently imaged at 1,000× magnification using an Axio Imager A2 Zeiss microscope equipped with a GFP filter set. Images were acquired using the ZEN 2012 (blue edition) software. The bacterial cell diameters were determined using the Fiji software. Only nondividing cells (cells without any obvious fluorescent dots or lines at the mid‐cell), were used for cell diameter measurements. The cell diameters of 50 cells were measured and the average cell diameter determined. The experiment was conducted three or four times (as indicated in the figure legend) and the averages and standard deviations of the average cell diameters plotted.

To determine the subcellular localization of the fluorescent SEB-1-sfGFP protein under heat stress, 200 μl of a conidial suspension (2 × 10⁶ conidia/ml) from the Δseb-1 complemented strain were inoculated onto coverslips, covered with VM liquid plus 1% sucrose, and incubated at 30° for 10 hr. After incubation, the coverslips were transferred to fresh VM media containing 0.5% sucrose preheated to 45° and incubated at the same temperature for up to 2 hr. For osmotic stress, the coverslips were shifted to fresh VM media containing 0.5% sucrose, and either 1.5 M sorbitol or 1.5 M NaCl, and incubated at 30° for up to 4 hr. To analyze oxidative stress, the coverslips were shifted to fresh VM liquid media containing 0.5% sucrose and either 500 μM paraquat, 100 μM menadione, or 25 mM hydrogen peroxide, and incubated at 30° for up to 2 hr. For nuclei analysis, mycelia were fixed [3.7% formaldehyde, 50 mM NaH₂PO₄ (pH 7.0), 0.2% (v/v) Tween 80], washed twice with PBS and stained with 100 µl DAPI (4’,6-diamidino-2-phenylindole, 0.5 mg/ml) for 5 min. Fluorescence was visualized using a fluorescence microscope with excitation and emission wavelengths of 359 nm and 461 nm, respectively. The images were captured using an AXIO Imager.A2 Zeiss microscope coupled to an AxioCam MRm camera and processed using AxioVision software, version 4.8.2.

Slides were documented and analyzed using an Axio Imager A2 microscope and Zen Blue software (Zeiss, Germany). The first field to be captured was selected at random near the luminal epithelium. Five nonoverlapping fields (20× objective) were documented per case. A positive cell was defined as an immuno-positive structure associated with a hematoxylin-stained cell nucleus. Cells were counted manually by two investigators unaware of the identification of the groups. The cell count tool from FIJI/Image J software (National Institutes of Health) was used. Cells within the glandular epithelium, blood vessels, or areas filled with blood were not considered. Data were expressed as mean number of cells/mm². The ratios between markers were calculated and expressed as percentages. Statistical analyses were performed using the GraphPad Prism software (Graphpad Software, Inc., San Diego, CA, USA). Mean values were evaluated by the nonparametric Mann–Whitney test. Correlations were determined by the Spearman correlation test, and frequency distributions were compared using the chi-square test. Values of p ≤ 0.05 were considered statistically significant.

Seven-day-old protonema tissues of KMR5045 were used to isolate protoplasts. Protonema were pre-incubated for 1 h in 0.5 M mannitol with slow rotation. Two experiments were performed to optimize the mannitol buffer and enzyme concentrations. The first experiment was designed to determine the optimal concentration of mannitol buffer. Three mannitol concentrations (0.4, 0.5, and 0.6 M) were tested in the isolation procedure. The second experiment was designed to compare the effect of enzyme concentration on protoplast yield and viability. Three concentrations (0.5, 1.0, and 2.0%) of driselase (Sigma-Aldrich, United States) dissolved in 0.5 M mannitol were tested. After induction of cell plasmolysis with mannitol, the cell suspension was treated with enzyme solution and incubated in darkness for 2 h at 20°C with constant gentle agitation on a rotary shaker. The digested cells were passed sequentially through 100 and 40 μm nylon sieves and the filtrate was centrifuged in a swinging-bucket rotor (124 × g, 10 min). The pellet was resuspended in 20 mL of 0.5 M mannitol and centrifuged again. After centrifugation, the pellet was resuspended in 0.5 M mannitol. The isolation yield of protoplast cells was determined using a counting chamber under a visible light Axio Imager A2 microscope (Carl Zeiss, Germany). The experiments were conducted with four biological replications.

A minimum of 10 plants were characterized for each mutant. Chromosome spreads were stained with DAPI and centromere FISH, and immuno-localization experiments were carried out as described previously [79 (link)]. Rabbit polyclonal AtRAD51 and γ-H2AX antibodies were used at 1:200 fold dilutions and Alexa Fluor 488 Goat Anti-Rat IgG (H+L) secondary antibody (A-21428, Invitrogen, Carlsbad, CA, USA) was used at a 1:1000 fold dilution [80 (link)]. Chiasmata distribution statistics were performed following the protocol of Sanchez et al. [81 (link)]. BAC DNA extraction (F19K16) and probe labeling were described previously [43 (link)]. Images of chromosome spreads were obtained using an Axio Imager A2 microscope (Zeiss, Heidelberg, Germany) equipped with a digital camera (Canon, Tokyo, Japan), and processed using Photoshop CS (Adobe Systems, Mountain View, CA). Images were initially captured in black & white and, if necessary, globally false-colored post-capture for visual contrast. AtRAD51 and γ-H2AX foci in WT and mutant lines were counted and statistically analyzed using ImageTool version 3.0 software (University of Texas Health Science Center, San Antonio, USA).
In mutants that lacked synapsis, we distinguished zygotene from pachytene chromosomes by their relative condensation, with pachytene being more condensed than zygotene chromosomes.