Lsm pascal

Cells were grown to approximately 80% confluence on coverslips; they were washed with phosphate-buffered saline (PBS), fixed with 4% PFA/PBS for 20 min at room temperature, washed three-times, permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature. After washing with PBS, nonspecific binding of the antibodies was blocked with 10% FBS/PBS for 30 min at room temperature. They were then stained for 1 h at room temperature with the rabbit anti-FLAG antibody diluted in 5% FBS/PBS, followed by an anti-rabbit AlexaFluor^® 488 (ThermoFisher Scientific) for one hour at room temperature. In the case of HBcAg detection, an AlexaFluor^® 594 conjugated secondary antibody was used for visualization. For specific staining of the ER and the Golgi, AlexaFluor^® 594 Concanavalin A and AlexaFluor^® 647 Lectin HPA (Helix pomatia agglutinin; ThermoFisher Scientific) antibodies were used respectively to stain the cells for a further hour at room temperature. For specific staining of actin and Tubulin, AlexaFluor^® 647 Phalloidin and a mouse a-Tubulin (Sigma-Aldrich) were used, respectively. For Tubulin, a secondary anti-mouse AlexaFluor^® 594 antibody was used for visualization purposes. Images were taken on an Axiovert 135 TV (Zeiss) fluorescent microscope or an EVOS FL Auto Cell Imaging System (ThermoFisher Scientific), as well as a confocal microscope (LSM Pascal, Zeiss).

MCF-7 cells seeded on chamber slides were treated with 25 or 50 μg/mL GSE for 2 h or 24 h, and the related controls were used for immunochemistry and confocal microscopy analyses as described in [32 (link)]. Different experiments were done using both anti-Cx43 from Sigma-Aldrich and from Zymed as primary antibody (1:250 in PBS-BSA). In addition, Alexa Fluor^® 633-conjugated anti-rabbit IgG or Alexa Fluor^® 488 conjugated anti-rabbit IgG as secondary antibodies, and PI or SYBRGreen (all products from Invitrogen) as nuclear counterstain were used. Only images obtained from immunodetection with anti-Cx43 from Sigma-Aldrich, Alexa Fluor^® 633-conjugated anti-rabbit IgG and SYBR green are shown. Negative controls were obtained with non-immune serum followed by a secondary antibody or with primary or secondary antibodies only. A minimum of five randomly selected areas from each chamber of each GSE treatment or control were analysed by confocal microscopy (LSM Pascal, Zeiss, Munich, Germany).

Preparation of COS-7 cells transfected with plasmid DNA, and confocal microscopic analysis with LSM Pascal (Zeiss) were performed as described [10 (link)]. Immunostaining was performed using mouse anti-Myc 9E10, mouse anti-HA 12CA5, rabbit anti-HA Y-11 (Santa Cruz), mouse anti-pan lamin (X67, X167, X233) (Abcam) and anti-Nup153 QE5 (Abcam) antibodies as primary antibody and Alexa Fluor 488-, Alexa Fluor 555-, and Alexa 546-conjugated antibodies (Molecular Probes) as secondary antibody. Nuclei were stained with SytoxGreen (Molecular Probes). For co-immunostaining with lamin, transfected cells are fixed in methanol at -20 °C.

Z-projections of cell image stacks were generated using ImageJ. βcat fluorescent intensity: Cells were outlined, mean intensity measured, background subtracted, and βcat average intensity of a transfected cell normalized to mean of the βcat intensity of 2–3 adjacent untransfected cells. 10 cells were each measured in 3 independent experiments. Puncta colocalization of APC2ΔSAMPs with Axin in BIO/LiCl treated cells were determined by scoring for puncta formation in the APC2ΔSAMPs channel. 100 cells were scored in 3 independent experiments. APC:Axin complex size: Particle Analyzer of ImageJ was used. Background was subtracted and threshold for particles set to 200. Cytoplasmic puncta of 10 cells were averaged. Cell images were taken with LSM Pascal (Zeiss) with a resolution of 5.7 pixel/μM. Mean number of particles per cells was calculated from size measurements. Puncta volumes were measured using Imaris Software (Bitplane, Concord MA) from image z-stacks acquired on the Deltavision OMX (GE Healthcare Life Sciences). For comparing sequences, ClustalW2 (EMBL, Germany) was used for alignment. Statistical tests used the Student's t-test.

β-Glucuronidase activity in the roots and shoots of ENA1 promoter::GUS transgenic plants was determined using a histochemical assay, as described previously (Inoue et al., 2003 (link); Nozoye et al., 2011 (link), 2015 (link)). Subcellular localization of ENA1 in onion epidermal cells or rice roots was observed as described previously (Nozoye et al., 2011 (link), 2015 (link)). FM-64 (1 mM; Molecular Probes) was used for staining the plasma membranes by incubation of rice roots section on glass slides at room temperature. Confocal images of rice roots were acquired with a laser scanning microscope (LSM Pascal and LSM 780, Zeiss, Germany) equipped with a 10×/0.45 M27 objective. Excitation/emission wavelengths of 488 nm/490–540 nm or ∼515 nm/640 nm were used for detection of GFP or FM4-64 fluorescence, respectively.