Axiocam mrm

The immunofluorescent images were taken with a ZEISS inverted fluorescence microscope (AxioCam MRm, Zeiss) under a 20 × or 40 × objective lens that was equipped with a monochromatic digital camera (AxioCam MRm, Zeiss). AxioVision Rel. 4.8 acquisition and processing software was used to acquire uniform digital images for each antibody throughout the experiments, and the images were converted to TIFF files. ImageJ and Adobe Photoshop were used to analyze and manage the TIFF files.
As the present study was mainly concerned with whether brain changes appeared among the W, M, N and St, the brain levels (from A6.2-A10.2 in the chick brain atlas) containing the above regions were chosen. There were no significant differences among these brain levels, and the results reported were combined.
The obtained values (the numbers of labeled cells) were compared by using the SPSS 11.5 software package. One-way ANOVA was conducted to compare the differences among the studied groups. Before ANOVA, the distributions of dependent variables were tested for normality, and homogeneity of variance was assessed for equality of error variance (Levene’s test). The data are presented as the mean ± SEM. Differences between the groups of data were considered either nonsignificant (p > 0.05) or significant at various levels (*p < 0.05, **p < 0.01 and ***p < 0.001).

Cell uptake of PLGA-CDs1% NPs and PLGA-CDs20% NPs (0.5 mg mL⁻¹) was evaluated by fluorescence microscopy (Axio Cam MRm, Zeiss, Oberkochen, Germany) on MDA-MB-231 after 4, 6 and 24 h of incubation. Cells were seeded in an 8-well plate at a density of 1 × 10⁴ cells per well (200 μL) and growth in DMEM. After 24 h the medium was replaced with equal volume of fresh medium containing the nanosystem (0.5 mg mL⁻¹). After different incubation time, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) washing cells with DPBS pH 7.4, adding the DAPI solution (100 μL) and incubating cells for 10 min. Cells were then washed up with fresh DPBS (200 μL three times) and images were recorded by a fluorescence microscope using a Zeiss Axio Cam MRm (Zeiss AG, Oberkochen, Germany).

Live cell images were acquired using a Zeiss AxioObserver D1 inverted fluorescence microscope with AxioCam MRm and a 10× objective. Filter excitation (EX) and emission (EM) bandpass specifications were as follows (in nm): GFP (EX: 470/40, EM: 525/50); mCherry (EX: 545/25, EM: 605/70). Immunostaining images were acquired using the previous Zeiss AxioObserver or the Zeiss AxioImager A1 upright fluorescence microscope with AxioCam MRm and 10x or 20x objectives. Filter excitation (EX) and emission (EM) bandpass specifications were as follows (in nm): DAPI (EX: 365, EM: 445/50); FITC (EX: 450/90, EM: 515/65). For phalloidin immunostaining, cells were seeded on sterile coverslips or in culture plates directly and treated with 1 μg/mL tetracycline for 24 h before fixation with 4% paraformaldehyde. After fixation, cells were permeabilized with 0.1% Triton X-100 and incubated with 0.1 μg/mL FITC-conjugated phalloidin (Sigma) for 1 h at room temperature. Next, cells were stained with 300 nM 4′,6-Diamidino-2-Phenylindole (DAPI) and mounted on glass slides with Vectashield Hardset mounting media (Vector) for fluorescence microscope imaging, when not imaged directly in wells.

Tissues and cells were viewed with a Nikon C1 confocal microscope. S2 cell images were obtained using a Plan Apochromat 60×/1.45 NA oil immersion objective (Nikon). Salivary gland images were obtained using 20×/0.75 NA objective lens. Images were captured and analyzed with EZ-C1 Ver 3.00 software. Alternatively, tissues and cells were viewed using an Axioplan 2 microscope (Zeiss, NY, USA) with ApoTome (Zeiss) and imaged with a Zeiss AxioCam MRm. Images were captured and analyzed using AxioVision software. For S2 cell images and salivary gland images, a 64× lens and 20× lenses were used respectively. For quantification of tubulin network phenotypes, 10 salivary glands each from wild type and mutant were analyzed, with two images/gland captured using the ApoTome and Zeiss AxioCam MRm camera. Cells displaying normal, fragmented, sparse and dense tubulin networks were manually counted. For Z-stack series, images were captured using 40× lenses at 2 µm thickness.

Differential interference microscopy (DIC) and fluorescent images were visualized with a Zeiss Axio Imager fluorescence microscope (64X objectives). Images were taken with an AxioCam MRm digital camera with ZEN Pro software (Zeiss). The same exposure time was used to image for every strain analyzed in one data set. Images were analyzed using ImageJ/Fiji software. For indicated strains a DAPI stain (NucBlue, Invitrogen) was used to visualize the nucleus. DAPI stained cells were analyzed using the DAPI stain, Cel1-GFP were visualized using the GFP channel. Cells sizes were measured using the ImageJ measurement tool. Capsule thickness was calculated using the equation:
$$capsulethickness=\frac{\left(celldiameterincludingcapsule-cellbodydiameter\right)}{2}$$
Histopathology images were taken with a color camera (AxioCam MRm) attached to the Zeiss Axio Imager and analyzed using ImageJ/Fiji.