Axioplan 2 microscope

Bright-field images were taken using a Zeiss Axioplan 2+ microscope, Nikon Digital Sight DS-F12 camera, and Nikon NIS 4.0 imaging software. Images of immunohistochemistry and immunostaining of electroporated brains and dissociated cultures were obtained using a Zeiss LSM 5 Exciter–AxioImager M1 imaging system and Zeiss LSM710 imaging system. Image stacks were generated by scanning at intervals of 0.5–1 μm using filters of the appropriate wavelengths. The stacks were analyzed, merged, and projected using ImageJ software (RRID: SCR_003070) from the National Institutes of Health. Figure panels were prepared using Adobe Photoshop CS6. Figure 2C,D shows stitched composites from multiple confocal image frames.

Bright-field images were taken using a Zeiss Axioplan 2 + microscope, Nikon Digital Sight DS-F12 camera, and Nikon NIS 4.0 imaging software. Images of immunohistochemistry were obtained using a Zeiss LSM 5 Exciter-AxioImager M1 imaging system and Zeiss LSM510 imaging system. Image stacks were generated by scanning at intervals of 4 μm for lower magnification and at intervals of 0.8 μm for higher magnification using filters of the appropriate wavelengths. The stacks were analyzed, merged, and projected using ImageJ software from the National Institutes of Health. Figure panels were prepared using Adobe Photoshop CS6.

Multidirectional chromosome painting was performed using whole chromosome probes from CBR and PHA (Phyllostomidae), generated by flow cytometry [13 (link)]. The probes were amplified and labeled by DOP-PCR [19 (link), 20 (link)] and hybridized following procedures previously described [13 (link), 20 (link)]. Briefly, the slides were incubated in a pepsin enzyme solution, washed in 2× SSC and dehydrated in an alcohol series (70%, 90% and 100%). Subsequently the slides were oven aged at 65 °C for 1 hour, denatured for 1 min in formamide solution (70% formamide/ 2× SSC) and incubated in hybridization solution (14 μl of solution containing: 50% formamide, 2× SSC, 10% dextran sulfate, 5 μg of salmon sperm DNA, 2 μg mouse Cot-1 DNA and 1 μl of labelled PCR product) for 3 days. After post-hybridization stringency washing, biotin-labeled probes were detected with avidin-Cy3 or avidin-FITC (1 μg/ml; Amersham). For dual color-FISH we used both biotin-labeled and direct-labeled probes. Digital images were captured using Axiovision 3.0 software via an Axiocam mRM CCD camera, coupled to a Zeiss Axioplan 2 microscope or with the Nis-Elements software on a Nikon H550S microscope. Chromosomes were identified by chromosomal morphology and by staining patterns with the fluorochrome DAPI (4 ‘, 6-diamidino-2-phenylindole).

After 42 h of fixation in 10% chilled formalin solution, the intestines were dehydrated in increasing gradients of ethanol (50%, 65%, 75%, 85%, 95%, and 100%). The samples were embedded in hydroxyethyl methacrylate (Historesin mounting medium, Leica, Germany) for three days. A series of ~5–10 histological sections were performed on each sample (section thickness ~5 µm), mounted on slides, and stained using Haematoxylin and Eosin (H&E; Sigma Aldrich, St. Louis, MO, USA). Image acquisition was performed using a Zeiss Axioplan 2 microscope mounted with a Nikon (Tokyo, Japan) digital camera DXM 1200F. The external circumference of the serosa, mucosal height, and muscularis layer thickness were quantified using ImageJ (Fiji) software, version IJ 1.46r, Wisconsin, USA, [36 (link)]. The number of goblet cells was determined by manually counting them in the complete mucosal region under microscope.

Fixed mid-gut and posterior liver tissues were dehydrated in an ethanol gradient (50%, 60%, 70%, 85%, 90% and 96%). Dehydrated tissues were embedded in hydroxyethyl methacrylate (Historesin^®, Leica, Heidelberg, Germany) and sectioned into a series of 30 sections per sample (section thickness ~4 μm). Sections were mounted on slides and stained using haematoxylin-eosin (H/E). To ensure that H/E could be used for counting of goblet cells, Periodic Acid-Schiff and Alcian blue staining were performed on other sections of the same sample for verification (S2 Fig).
Microscopic analyses were performed using a Zeiss Axioplan 2 microscope mounted with a Nikon digital camera DXM 1200F. For mid-gut and posterior liver analysis, nine samples were collected from each group (total of 36 samples). For every gut sample, ten cross-sections were quantified for external circumference of serosa (ECS), mucosal height (MH) and muscularis layer thickness (MLT). Hepatocyte diameter (HD) was quantified by taking thirty measurements per liver section. All measurements were performed using the Fiji software [39 ]. The goblet cell number (GCN) was determined by counting them in the entire mucosal region under the microscope. Each sample was assessed twice by two researchers and averaged before using it as a representative measurement.