Axiocam mrm camera

Fluorescence imaging was performed on live animals. Briefly, animals were immobilized using 30 mg/ml 2,3-butanedione monoxamine (Sigma-Aldrich) prepared in M9 as previously described (Sieburth et al., 2005 (link)). In order to visualize and image, the animals were immobilized with 30 mg/ml BDM on 2% agarose pads (prepared in M9); 1 μm (total depth) Z-series stacks were collected using a Carl Zeiss fluorescence microscope Axio Imager Z2 with the Axiocam MRm camera equipped with GFP and red fluorescent protein filters. Images were collected and analyzed using the FIJI image J software (Schindelin et al., 2012 (link)). Maximum intensity projections of Z-series stacks were used for the analyses of expression and localization of fluorescent markers. The exposure settings were kept identical for all images taken in a single experiment. Imaging was done in the head region of the animals and 25–30 animals were imaged for each experiment. To image AWC and ASI cilia, imaging was performed using the Leica TCS SP8 confocal microscope, using the argon laser at 10% gain.

A Weisner reaction was carried out to histochemically locate the lignin according to the standard protocol (Mitra and Loque, 2014 (link)). The fixed samples were dehydrated in a graded ethanol series and finally in a xylene series. Fresh hand-cut sections (~20 µm) of PMW-2016-1 and DPW-621-50 culms were incubated in phloroglucinol solution (2 volume 3% in ethanol:1 volume concentrated HCl) for 2 min. For toluidine blue staining, 0.02% aqueous solution of toluidine blue O was used to stain the free-hand sections for 2 min (Li et al., 2003 (link)). The sections were rinsed with distilled water 3-4 times until the solution was clear. These sections were then mounted with DPX and observed under a bright-field microscope. Sections were viewed under magnification of 10X and 40X for vivid visualization of lignin distribution across the stem and to differentiate the major tissues. Digital images were scored/taken using an Axiocam MRm camera.

M. pneumoniae cells were grown in a 75 cm² flask in Hayflick medium for 4 days under standard conditions. After 4 days, the medium was removed and cells were re-suspended in 5 ml of fresh Hayflick medium, and subsequently scrapped and collected. Aggregates were then removed by passing the cells through a syringe with a G25 needle (10 × ) and a 0.45 μm filter, and mixed with 5 ml of 6% gelatin. Then, cells were grown on borosilicate coverglass slides (Thermo Scientific) for 6 h. Cells were fixed in a final concentration of 4% formaldehyde (Pierce) for 20 min at RT followed by 40 min at 4 °C, before further fixing with cold methanol at −20 °C O/N. DAPI was added on the slides (10 μg ml⁻¹) for 15 min and three washes were done with PBS.
The cells were observed on Axio Observer Z.1 equipped with a × 63 1.4 Oil objective and an Axio-Cam MRm camera. The fluorescence intensities of DAPI were measured by using the command ‘analyse particles' using Fiji and taken as measurements of the DNA contents of individual cells.

Strains were grown at 30 °C, washed in water to remove trace media constituents and re-inoculated into fresh YPD medium supplemented with alcohols to OD₆₀₀ 0.1 then incubated at 37 °C. As positive and negative controls, cells were re-inoculated into 10% serum medium or YPD lacking alcohol, respectively. At various times during incubation (1 h, 2 h, 3 h, 4 h), the morphology of the cells was monitored using a Nikon Eclipse E600 and Axiocam MRm camera. Images were acquired using Axiovision 4.5 software. The proportion of cells forming germ tubes or pseudohyphae were counted using a haemacytometer. Each count was repeated three times and the mean of the three counts recorded. Colony morphology on solid media was assessed by plating on YPD agar or YPD agar supplemented with the indicated alcohol, 10% serum or cycloheximide concentrations ranging from 10 μg/ml to 200 μg/ml. Micro-colonies were photographed using a Nikon Eclipse E600 and Axiocam MRm camera and the images were acquired using Axiovision 4.5 software.

The rescue assays shown in Figures 2 and 4 were done as follows. About 60,000 U2OS cells were plated on a coverslip in a 12-well dish 24 h before transfection. For each sample, 50 nM of the respective siRNA was incubated with 500 ng of the respective DNA in 50 μl of Opti-MEM for 5 min. At the same time, 2 μl of Lipofectamine 2000 (Invitrogen) was diluted into 50 μl of Opti-MEM. After 5 min, the diluted Lipofectamine was added to the siRNA/DNA and mixed gently. The mixture was left to incubate for 20 min at room temperature (RT). Each 12-well was replaced with 900 μl of fresh media. The siRNA/DNA/Lipofectamine mixture was added to the well gently. The media was replaced 12 to 16 h later. The cells were fixed 48 h after the initial transfection. The samples were then processed for immunofluorescence as described above and imaged on a Zeiss Axiovert Observer D1 with a 63×/1.4 oil immersion lens and an AxioCam MRm camera. A minimum of 100 transfected cells were imaged for each sample for each replicate. Cells with two or more holes of 3 μm or greater were considered to have the hole-depletion phenotype and considered not rescued. The four relevant plasmids, NOMOr-FLAG, Climp63-FLAG, Alt2-FLAG, and Atl2K107A-FLAG, were blinded by a fellow laboratory member before transfections occurred. This experiment was repeated four times for each siRNA and plasmid combination.