Leica tcs sp8 x confocal laser scanning microscope

The full-length coding sequence of OsMYB7 subcloned into the entry vector pCR™8/GW/TOPO^® (Invitrogen) was transferred into the Gateway-compatible plant destination vector pEarleyGate 104 (Earley et al., 2006 (link)) by the LR reaction. The resulting vector was introduced into onion (Allium cepa) epidermal cells by particle bombardment with a Biolistic PDS-1000/He instrument (Bio-Rad, Hercules, CA, USA). After bombardment, the onion epidermal layers were incubated on Murashige and Skoog (MS) medium (pH 5.7) for 16 h at 22°C in the dark. The nuclei were then stained with 300 nM of 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) in phosphate-buffered saline (PBS) for 3 min in darkness before observation. Fluorescence emission was analyzed using a Leica TCS SP8 X confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) with the following parameters: excitation at 458 nm and emission at 514 nm for YFP, and excitation at 405 nm and emission at 488 nm for DAPI.

Validation of GCaMP3 expression in the target root cell types was conducted with an inverted Leica TCS SP8-X Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany; http://leica-microsystems.com). Seedlings from the Conviron were secured in a horizontal orientation on the stage of the microscope. Seedling roots were illuminated with the 488 nm line of the Argon laser using a 40× (numerical aperture 1.1) water immersion objective and emitted light detected at 510 nm. For some lines, single optical images were acquired at a pixel resolution of 1024 × 1024. For other lines, a Z-series was acquired by capturing 81 images at 0.4 µm intervals and 3-D images were generated using the LAS visualization software of the Leica confocal microscope.

Immunofluorescence (IF) analyses were performed as described [25 (link)]. HER2 was analyzed in 4 μm formalin-fixed, paraffin-embedded (FFPE) slices of the GHEA cohort using rabbit anti-human HER2 antibody (1:50) (Dako, Glostrup, Denmark) after antigen retrieval in 10 mM citrate buffer, pH 6.0, for 6 min at 121 °C, followed by the use of anti-rabbit AlexaFluor 546 secondary antibody (1:1000, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) Prolong (Thermo Fisher Scientific). Images were acquired on a Leica TCS SP8 X confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). DAPI was excited using a diode laser and detected from 410 to 458 nm, and Alexa Fluor 546 was excited by selecting a 553 nm laser line and detected from 557 nm to 667 nm. Nine images were acquired for each slide using an HC PL APO CS2 40X/1.30 oil-immersion objective, and a pinhole was always set to 1 Airy unit. Data were analyzed using a Leica LAS X rel. 3.1 software (Leica Microsystems GmbH). To avoid fluorescence signal saturation in tumors with the highest HER2 expression, images of all samples were acquired using the microscope setting parameters of the most positive tumor, and protein expression was scored as the mean fluorescence intensity (IF-score) in nine fields of each tumor slide analyzed with a 40x objective.