Inverted tcs sp8 confocal scanning laser microscope

Green fluorescent protein expression was monitored in seedlings of the above transgenic lines of A. thaliana germinated on control (Ctrl) and on Cd containing medium (25–50 μM respectively) and sampled at 8 DAG. Confocal images of median longitudinal sections were obtained using a Leica inverted TCS SP8 confocal scanning laser microscope, with a 40x oil immersion objective. The detection of Green Fluorescence Protein (excitation peak centered at about 488 nm, emission peak wavelength of 509 nm) was performed by combining the settings indicated in the sequential scanning facility of the microscope. Three independent replicates were performed and a minimum of 40 seedlings was analyzed for each sample.

Arabidopsis thaliana seedlings germinated on control (Ctrl) and on Cd containing medium (25–50 μM respectively) and grown in a vertical position were used. Three independent replicates were performed and for each sample, a minimum of 70 seedlings was analyzed. Data were statistically evaluated by Student’s t-test.
Root length was monitored until 21 days after germination (DAG) by scanning the plates and analyzing the resulting images through the open source processing program ImageJ¹.
For the meristem size analysis, seedlings at 8 DAG were stained with propidium iodide following the MPS-PI-staining protocol (Truernit et al., 2008 (link)). Confocal images of median longitudinal sections were obtained using a Leica inverted TCS SP8 confocal scanning laser microscope, with a 40x oil immersion objective, and excitation and emission wavelength were 600 and 640 nm, respectively. For meristem size evaluation, the distance and the number of cortex cells in a file extending from the QC to the first elongated cortex cell were measured (Dello Ioio et al., 2007 (link); Perilli and Sabatini, 2010 (link)). Three independent replicates were performed, and for each sample a minimum of 70 seedlings was analyzed. Data were statistically evaluated by using one-way ANOVA with Tukey post hoc test (P ≤ 0.05) after Shapiro–Wilk normality test.

Seeds of Arabidopsis transgenic lines (background Columbia 0), and in particular, the synthetic reporter pDR5::GFP, and different auxin transport proteins pPIN1::PIN1-GFP, pPIN2::PIN2-GFP, pPIN3::PIN3-GFP, pPIN4::PIN4-GFP, and pPIN7::PIN7-GFP, were germinated and grown as previously reported. Five seedlings (4 d old) were then transferred to a single Petri dish containing the same medium previously described and enriched with 50 μM protodioscin for each treatment and replicate (N = 6). The transplanted seedlings were then placed in a growth chamber for 6 d and grown as previously described.
The Arabidopsis roots were then collected, fixed for 1 min in 4% (w/v) paraformaldehyde in 1X Phosphate Buffer Saline (PBS) (pH 7.0) and mounted in a 1:1 solution of glycerol: PBS (1X). Confocal images of median longitudinal sections were acquired using a Leica inverted TCS SP8 confocal scanning laser microscope, with a 40 × oil immersion objective. The detection of Green Fluorescent Protein (GFP) (excitation peak centered at about 488 nm, an emission peak wavelength of 509 nm) was performed by combining the settings indicated in the microscope’s sequential scanning facility. More than 20 seedlings were analyzed per treatment, and four independent experiments were carried out.

E16.5, E18.5 mice brains were fixed with 4% praraformaldehyde (PFA)/PBS at 4 degrees overnight, and sequentially replaced to 15% sucrose/PBS and 30% sucrose/PBS at 4 degrees for cryo-protection, then embedded in OCT compound. Frozen brains were sliced into 80 μm thick sections on a cryostat (CM3050S, LEICA). Brain sections and whole embryos fixed with 4% PFA/PBS were subjected to floating or whole-mount immunohistochemistry. These samples were washed three times with PBS at room temperature (RT), incubated with 0.2% Triton X-100/PBS at R.T., blocked with 1.5% normal donkey serum (NDS)/PBS at RT, and then incubated overnight at 4 degrees with primary antibodies or Alexa-dye conjugated phalloidin in 1.5% NDS/PBS followed by incubation with Alexa 488, 555, or 647 conjugated donkey secondary antibodies (1:1000) in 1.5% NDS/PBS. Images of immunostained specimens were collected by BZ-X700 (KEYENCE), fluoscence microscopes Axioplan 2 (Zeiss), or an inverted TCS SP8 laser scanning confocal microscope (LEICA) at The Rockefeller University Bio-Imaging Resource Center.