Axiocam 506 color camera

Germbands were imaged with an AxioCam MRc camera (Zeiss) mounted on a Zeiss Axio Imager M1. Picture of germbands were taken with a 20X objective requiring image merging with Adobe Photoshop. Blastoderms were imaged in PBST using an Axiocam 506 color camera mounted on a Zeiss Discovery.V12 SteREO dissecting microscope. SYTOX-stained embryos were imaged using an Axiocam mono camera mounted on Zeiss Discovery.V12 dissecting microscope in the dark.

The Arabidopisis thaliana was treated by spraying CdTe-QDs (0.8 mmol/L), and the transport and distribution of QDs in the Arabidopisis thaliana organs was observed using laser confocal 2 days later. The samples were observed using a confocal (Olympus FV1000) microscope with 1.4 NA and 60× oil immersion objective. CdTe-QDs were visualized using 488 nm laser excitation and 500 to 550 nm spectral detection. Images were collected at a size of 512 × 512 pixels by using the FV10-ASW 4.2 viewer software. Pollen tubes stained with aniline blue and pollens stained with Alexander dye were observed with fluorescence microscopy (BX53, Olympus, Tokyo, Japan). Images were acquired using an Olympus DP71 CCD camera. Flowers, siliques and seeds were observed with a Zeiss Axio Zoom V16 Materials Stereo Zoom Microscope, and images were acquired using Zeiss Axiocam 506 color camera. Seeds within the silique were observed using an Olympus BX53 fluorescence microscope, and images were acquired using an Olympus DP71 CCD camera. Adobe Photoshop CS5 was used to splice the collected images.

Preliminary IHC tests with different anti-GD2 antibodies (Table 4) were performed on neuroblastoma and melanoma histological specimens and cell lines (data not shown), in order to set up the procedure. After extensive testing, the selected antibody was an anti-GD2 rabbit polyclonal antibody (1:200 dilution; Matreya LLC, Pleasant Gap, PA). Formalin-fixed, paraffin-embedded 4-μm-thick tissue sections from BC specimens were transferred to microscope slides. Sections were dried overnight at 40°C; after heat-induced antigen retrieval, slides were incubated with the anti-GD2 antibody for 12 minutes at 37°C, using an automatic immunohistochemical staining device (Benchmark XT; Ventana Medical Systems, Tucson, AZ). The reaction was subsequently detected with the appropriate reagent from Kit Ventana DAB (Ventana Medical Systems, Tucson, AZ). Lastly, sections were counterstained with Harris hematoxylin and analyzed by standard light microscope (Axio Imager M2 - Zeiss) with Plan-Apochromat 20X/0.8, EC Plan-Neofluar 40X/0.75 and EC Plan-Neofluar 63X/1.25-Oil objectives. Slides stained with no primary antibody were used as negative control. Photomicrographs were acquired with Axiocam 506 color camera (software ZEN pro - Zeiss).

Each slide was scanned for microscopic metastases, micrometastases and TP-3 positive single cells. Micrometastases were defined as clusters of ≥ 5 and ≤ 50 TP-3 positive cells. Clusters > 50 cells were defined as microscopic metastases, while clusters of < 5 TP-3 positive cells were defined as TP-3 positive single cells.
IHC stained slides were evaluated using a Zeiss AX10 microscope, equipped with a Zeiss axiocam 506 color camera, coupled with Zen pro 2012 (blue edition) image acquiring software (Carl Zeiss Microscopy GmbH, Jena, Germany). The total number of microscopic metastases and micrometastases, if present, were counted in the entire slide for each sample. The number of TP-3 positive single cells was counted in 10 high-power fields (HPF, defined as one field at 400x, equivalent to 0.196 mm² for the microscope used). Areas with folded up tissue were excluded from the analysis. Slides where staining was too weak to identify positive cells or significant unspecific staining was present, were also excluded.

Cells were observed under an Axio Imager D2 phase contrast microscope (Zeiss, Germany) and photographed using an Axiocam 506 color camera (Zeiss, Germany). To monitor the growth rate of cells after RPB1 RNAi, the number of cells was measured every day before feeding for a period of seven days and compared with the control silencing (see Supplementary Data 1).
To evaluate the efficiency of ND7 RNAi, cells were treated with 20% acetic acid to assess their ability of discharging trichocysts. By comparing with the control, cells were divided into three categories: almost all trichocysts discharged, a few trichocysts discharged, and no trichocyst discharge. The number of cells in each category were then calculated (see Supplementary Data 1).

ISF (30 ml/kg) or control solution (30 ml/kg) was po administered in C57BL/6J mice and those systemically treated with capsaicin, both fasted 16 h. At 30 min after injection, mice were transcardially perfused with Zamboni solution (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 7.4) under anesthesia. The nodose ganglia (NGs) and brain were collected, postfixed in the same fixative for 2 and 4 h at 4°C, respectively, and incubated in phosphate buffer containing 30% sucrose for 48 h at 4°C.
Longitudinal sections (10 µm) of NGs were cut with 60 µm intervals using a precision cryostat (Leica Microsystems, IL). Coronal sections (40 µm) of the hindbrain were cut with 120 µm intervals using a freezing microtome (Yamato Kohki industrial Co. Ltd., Saitama, Japan). Rabbit polyclonal antibody against phospho-p44/42 MAPK (Thr202/Tyr204, pERK1/2) (1/500; #9101; Cell Signaling Technology, MA, USA) and Alexa 488-conjugated goat anti-rabbit IgG (1/500; A11008; Invitrogen, CA, USA) were used for detection of pERK1/2 in NG and medial NTS. Fluorescence images were acquired with AxioObserver Z1 microscope and Axiocam 506 color camera (Zeiss, Oberkochen, Germany). Neurons immunoreactive to pERK1/2 in medial NTS (bregma −7.32 to −7.76 mm) were counted an averaged per mouse. In NGs, a number of pERK1/2-positive NG neurons in four sections per mouse were counted and averaged.