Lsm 5 image browser

For image acquisition, a Zeiss LSM 510 meta laser scanning microscope using the 488nm excitation line of the argon laser was used. Fluorescence was detected using a 488/543 nm dichroic beam splitter and a 505-530nm band pass filter for Alexa-Fluor 488. Images were acquired using an oil immersion objective; Plan-Apchromat 63X (1.4 numerical aperture). Post-acquisition image processing and Z-stack image projections were processed using LSM 5 image Browser (Zeiss, Cambridge, UK).

The intact coding region sequences of JcFATA and JcFATB without a stop codon were amplified by RT-PCR with the primer pairs of JcFATA-PGFP-F and JcFATA-PGFP-R and JcFATB-PGFP-F and JcFATB-PGFP-R, and inserted into the transient expression vector pUC18-35S-eGFP between the CaMV 35S promoter and the GFP (green fluorescent protein) gene, generating an in-frame fusion for each gene. The leaves of 8-day-old Arabidopsis seedlings were cut into 1–2 mm pieces using a fresh sharp blade and used for protoplast preparation. Protoplasts were quantified using a hemocytometer under a microscope, and the fusion constructs for each gene and the control expression vector was introduced into the protoplasts as described [93 (link)]. GFP signals were observed under a fluorescence microscope OLYMPUS MF30 (Olympus Corporation, Tokyo, Japna) with the excitation and emission filters Ex480 ± 20/DM505/BA535 ± 25 and Ex535 ± 25/-DM565/BA645 ± 37.5 for GFP and chlorophyll auto-fluorescence, respectively. All fluorescence images obtained were processed with LSM 5 Image Browser (Carl Zeiss AG, Oberkochen, Germany).

CHO cells transfected or not with pValac::dts::IL-4 and pValac::dts (negative control) were fixed with 2 % paraformaldehyde; were permeabilized with 1 % Triton X-100 (Sigma Aldrich); and were stained with an anti-IL-4 primary antibody [IL-4(NYRmIL-4) (Santa Cruz Biotechnology, Inc.)], a secondary antibody conjugated to the Alexa Fluor 488 fluorophore [Alexa Fluor 488 rabbit anti-rat IgG (H + L) conjugate (Life Technologies)], and 4′-6-diamidino-2-phenylindole (DAPI) (Life Technologies), as recommended by the manufacturers.
Images were captured with the Zeiss LSM510 META confocal microscope (Zeiss). The argon laser and filter set 09 were used with an emission wavelength greater than 510 nm to detect the Alexa Fluor 488 fluorophore (Life Technologies), while the epifluorescence and filter set 01 were used to detect DAPI (Life Technologies). All the images were visualized using LSM 5 Image Browser (Zeiss) software.

The functional resorption activity of the differentiated osteoclasts was evaluated by resorption pit formation, as described below. Cells were cultured for 30 days on top of dentine disks (Immunodiagnostic Systems, Boldon, UK) in 96-well culture plates in the presence of M-CSF (20 ng/mL), RANKL (50 ng/mL), or IL-7 (2 ng/mL) to differentiate osteoclasts. Then, the dentine slices were washed three times and immersed in 70% sodium hypochlorite to remove adherent cells. The resorption lacunae were counterstained with 1% (w/v) toluidine blue in 0.5% sodium borate for 60 s (Sigma-Aldrich). Photographs were taken through an LSM 5 PASCAL confocal microscope (Carl Zeiss, Jena, Germany) to analyze the surface topography and the area of the resorption pits was measured in four randomly selected areas for each dentine slice with the LSM 5 Image Browser (Carl Zeiss). Roughness parameters obtained were:roughness average (Ra), which is the main height calculated over the entire measured length or area, Rq, statistical moments of peak distribution (symmetry), Rz, mean roughness depth, and Rv, maximum profile valley depth, which are the distances from the mean line/surface to the highest/lowest point in the evaluation length/area (26 (link)).

WT or GCN2^−/− MEFs were infected with DENV at moi 3 and incubated for indicated time points (as indicated in the results section). Post incubation, the infected cells were fixed with 4% paraformaldehyde in 1X PBS for 15 min followed by permeabilization with 0.2% Triton-X 100 for 20 min at room temperature. The cells were further blocked with 10% FBS or 1% BSA for 1 h at room temperature. Following washes with 1X PBS, cells were incubated with primary Ab for 2 h at 37°C followed by incubation with fluorescent secondary Abs, Alexa Fluor-488 (Invitrogen) at 37°C for 1 h. Next, cells were washed three times with 1X PBS and immunostained cells were finally mounted using Prolonged Gold Anti-fade Reagent containing DAPI (Invitrogen) or Vectashield Mounting Medium with DAPI (Vector Laboratories). The primary antibodies used were goat anti-COX2 (Santa Cruz), mouse anti-Dengue 1, 2, 3, 4 (GeneTex), mouse anti dsRNA-J2 (English and Scientific Consulting Kft.), and mouse anti-p65 (Santa Cruz). The immunofluorescence images were captured under 63X oil based objective using a LSM510 confocal microscope (Zeiss). Further, the images were processed and analyzed in Zeiss LSM5 image browser. The analysis of intensity profiles was carried out using Fiji/Image J software (NIH).

Cells were fixed in a mixture of 4% (w/v) paraformaldehyde in PBS (pH 7.4) overnight at 4 °C. After three washes in PBS buffer, cells were incubated with Bodipy 493/504 (Thermo Scientific, Grand Island, USA) at a final concentration of 10 µg/ml in PBS buffer for 1 h at room temperature. This was followed by the washing of cells three times in the same buffer and re-suspended in ProLong Gold anti-fade reagent (Thermo Fischer Scientific, Waltham, USA). Samples were then analyzed with a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Jena, Germany) using a 63× Plan-Apochromat 1.4 NA oil-immersion lens. An argon laser was used for Bodipy 493/504 and chlorophyll excitation and the emission spectra were collected in two separate channels, 500–515 nm and 630–670 nm, respectively. Z-series images were collected and processed with the LSM 5 Image Browser (Carl Zeiss, Jena, Germany). LD morphometrics were performed by the same software using 3D reconstruction confocal images of 70 cells for each analyzed line.