Dp26 camera

Digital bright field images were acquired using Olympus BX46 microscope equipped with DP26 camera and Labsens standard software (Olympus). Immunofluorescence images were acquired using Leica SP II confocal microscope and Leica Application Suite (Leica Microsystems B.V.). Image cytometry analysis of DAPI fluorescence intensity was done by using cell profiler software available at http://cellprofiler.org/ and previously described [18 (link)]. Quantification of binucleation using immunofluorescent and e-cadherin stained images obtained from three animals per genotype was done as previously described [8 (link)]. Total numbers of binucleated exocrine cells were counted under 63x objective and 1.5 zoom factor using immunofluorescent stained images. Percentage of binucleation was calculated by dividing total binucleated cells to the total number of cells counted in the field multiplied by 100%. Data are presented as average of five counted fields per genotype. Percentage of acinar cells with evidence of mitosis was obtained by using the formula: Total number of mitotic cells in 4–5 randomly selected high power fields (400x maginification) divided by 1000 multiplied by 100%. Other cell types such as duct cells, connective tissues and endothelial cells were excluded in the analysis.

The bulk was extracted from rice root following the method described by Barillot et al. (2013) (link). A modified Baermann tray technique was used for extracting nematodes from the bulk (Schindler, 1961 ). The rice roots were washed carefully under running water to eliminate all traces of dirt particles, meticulously chopped into small pieces (1–2 cm), and then blended in 0.8% NaOCl for 30 s. For 10 min, the mixes were left at room temperature before applying a modified Baermann funnel method (Hooper, 1986 ). Nematode suspensions derived from soil and root samples were obtained following a 48-h incubation period and subsequently subjected to inspection using a stereomicroscope. (Olympus SZ51; Tokyo, Japan). The nematodes without any stylets were identified as free-living nematodes. The identification of phytonematodes was conducted at the genus level through the analysis of morphological traits (Tarjan, Esser & Chang, 1977 ). The body size, stylet length, and vulva position were measured using the CellSens imaging software (V1.6) with an Olympus DP26 camera (Tokyo, Japan). Meloiodogyne species were identified using the perineal pattern of adult females (Hunt & Handoo, 2009 ) and species-specific primers (Mg-F3 5′-TTATCGCATCATTTTATTTG-3′ and Mg-R2 5′-CGCTTTGTTAGAAAATGACCCT-3′) as described by Htay et al. (2016) (link).

Pictures of living cells were acquired using an Olympus IX73 fluorescence microscope fitted with a 40 X objective (0.75NA UPlanFLN, Olympus) or a 20 × 0.40 NA objective (LCAChN 0.4NA, Olympus), a X-Cite Series 120Q lamp (Lumen dynamics), a DP26 camera (Olympus) and using the adequate filters set. For time series, cells were seeded in glass-bottom dishes (from MatTek or ibidi µSlide) in phenol red-free Leibovitz’s L-15 medium containing 10% FBS and pen./strep. For each time point, z-stacks were acquired using a Andor/Olympus Yokogawa CSU-X1 confocal spinning disk fitted with 60 X (UPLSAPO NA 1.35, Olympus) or 100 X (UPLSAPO NA 1.4, Olympus) objectives, a Andor iXon Life 888 EM-CCD camera and with temperature and humidity control. The white scale bars on representative pictures represent 10 µm.

Cells were seeded on glass coverslips (#1.5 thickness; ~ 170 µm, VWR). At the end of the treatment, the cells were washed twice with PBS, fixed by a 15 min incubation with 2% paraformaldehyde (PFA) in PBS and washed three times. The cells were then permeabilized 5 min with 0.2% Triton X-100 in PBS and washed three times with PBS. The coverslips were incubated 10 min in blocking buffer consisting in PBS 0.1% Tween-20 (PBS-T) containing 5% bovine serum albumin (BSA). The coverslips were incubated for 75 min with the primary antibodies diluted in blocking buffer (mouse anti-γH2AX antibody at 1/1000 and rabbit anti-53BP1 at 1/800), washed four times in PBS-T and then incubated 45 min with the secondary antibodies diluted in blocking buffer, washed four times in PBS-T and twice in PBS, incubated 15 min with 2 µg/mL DAPI (4′,6-diamidino-2-phenylindole) in PBS, washed twice with PBS, dipped in double-distilled water and mounted in VectaShield on a glass slide. Pictures were acquired using an Olympus IX73 microscope fitted with a 40 x UPlanFLN objective (Olympus), a X-Cite Series 120Q lamp (Lumen dynamics), a DP26 camera (Olympus) and using the adequate filters set. The white scale bars on each picture represent 10 µm.

Histochemical staining of in planta GUS (β-glucuronidase) activity was performed as described in Jefferson et al. (1987 (link)). After cocultivation, DR5::GUS seedlings removed from MS were immersed in GUS staining solution, which consists of 50 mM sodium phosphate (pH 7.0), 10 mM EDTA (pH 8.0), 2 mM K₄Fe(CN)₆, 2 mM K₃Fe(CN)₆, 0.1% (v/v) Triton X-100, and 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt). After applying vacuum for 5 min, they were incubated overnight at 37°C in the dark with gentle agitation (75 rpm). These seedlings were washed sequentially with 70, 80, and 90% ethanol solutions until all chlorophylls were removed and were stored in 90% ethanol at 4°C until imaging. Staining patterns were observed using a stereomicroscope (Olympus SZ60, Center Valley, PA, USA) and imaged using the Olympus DP26 camera.