A subset of electroporated specimens was used for quantitative analysis of axonal innervation and the sizes of the SAGs and sensory cristae. Immunostained cryosections were imaged with a 20x objective on a Nikon C1-plus confocal microscope. Maximal intensity projections were created and measurements were performed using NIH ImageJ software. NF70-positive pixels were summed to quantify sensory organ innervation as previously described (Fantetti et al., 2011 (link)). Briefly, alternate images containing NF70-positive axons were converted to black (neurites) and white (background). The innervated region was encircled, the image was cropped, and the number of black pixels was counted. Sensory organ size or SAG size (area in μm2) was measured by outlining either the Sox2-positive anterior/posterior crista or the defined edge of the SAG, respectively. The number of black pixels (neurites) per sensory organ, sensory organ size and SAG size was obtained for each section and then summed across the entire organ (typically 4-7 sections). Values for the right ear (electroporated) were normalized to the left ear (unelectroporated control). These ratios were calculated for the pEFX-GFP (control) and pEF1-Slit1-electroporated embryos and presented as mean ± standard error (SE).
C1 plus confocal microscope
Manufactured by Nikon
Sourced in Japan
The C1-plus confocal microscope is a versatile imaging system designed for high-resolution, three-dimensional visualization of biological samples. It features a laser-scanning confocal architecture that enables optical sectioning and the elimination of out-of-focus light, providing enhanced contrast and resolution compared to conventional widefield microscopy.
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6 protocols using c1 plus confocal microscope
1
Quantifying Sensory Organ Innervation
2
Immunostaining and Confocal Imaging of Hair Cells
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Images of immunostained sections and in situ specimens were taken with a Nikon Eclipse 800 equipped with a SPOT Flex digital camera (Diagnostics Instruments) and Spot 5.1 software. When appropriate, images of sections through the left ear and images of whole-mount left BPs were mirror-image flipped to facilitate comparisons with the right ear or the right BPs. Images of immunostained whole-mount BPs were taken with a Nikon C1-plus confocal microscope and a 60X lens. Image stacks were taken at the base (~25%), middle (~50%) and apex (~75%) along the BP (base was defined as 0% and apex 100%). For each HC in the field, the maximal cell perimeter was traced from the HCS-1 red channel without viewing the GFP fluorescence in the green channel, and the cross-sectional areas were calculated using ImageJ. Then each HC was viewed in the green channel and was categorized as either GFP-positive (GFP+) or GFP-negative for separate analysis. The number of ribbons per HC was counted as the number of CtBP2-positive foci. The numbers of GFP+ HCs and GFP+ supporting cells (SC) were counted in confocal image stacks using ImageJ and the ratios of GFP+ SC/HC were calculated. Student t-test and 2-way ANOVA was performed and graphs were made in Prism 6 software.
3
Visualizing Fly Reproductive Organs
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Digestive systems and ovaries from pharate female flies expressing GFP under the control of phm-GAL4 driver, and control flies were dissected on ice-cold phosphate-buffered saline and fixed in 4% paraformaldehyde for 2 h. Tissues were then washed and carefully mounted under Fluoromount-G mounting medium (Thermo Fisher Scientific) and stored in the dark until viewing. Images were taken on a Nikon C1Plus confocal microscope. Z-scans were obtained scanning preparations using a 20X objective and a z-step between 0.5 and 1.0 µm. Image projections were made using FIJI (58 (link)). Brightness and contrast adjustment were performed equally to all images using Photoshop CS5 (Adobe Systems).
4
Quantitative Confocal Microscopy Analysis
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Images were captured after satisfying the Nyquist criteria for sampling using a 100X, 1.49 NA TIRF objective on a Nikon C1 Plus Confocal microscope. Z Sections of 0.25–0.5 microns were taken. For every experiment, a series of test images were taken to identify exposure gains that minimized oversaturation and this gain was subsequently used for all conditions. 12 bit images were analyzed using Nikon Elements software. A single optical section at the plasma membrane/matrix interface was analyzed per cell. Fluorescence intensities were calculated as mean fluorescence intensity.
5
Immunocytochemical Analysis of Lens Epithelial Cells
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Human lens epithelial-B3 cells grown on glass coverslips were washed once with PBS (pH 7.4), fixed with 4% paraformaldehyde in PBS for 20 min, and permeabilized with 1% Triton X-100 for 10 min at room temperature. Non-specific antibody binding was blocked by incubation in PBS with 2% normal goat serum and 1% Triton X-100 for 1 h at room temperature. After fixation, permeabilization and blocking, cells were incubated overnight at 4°C with polyclonal antibodies (1:300, diluted in blocking solution) directed against human Cx43 (Invitrogen, Life Technologies, Carlsbad, CA, United States) or Cx46 (Santa Cruz Biotechnology) and monoclonal antibodies to αβ-crystallin (Santa Cruz Biotechnology) or Cx50 (Invitrogen, Life Technologies, Carlsbad, CA, United States). Cells were washed with PBS and incubated with goat anti-mouse IgG (H + L) secondary antibody; DyLight 488-conjugate and/or goat anti-mouse IgG (H + L) secondary antibody DyLight 594-conjugate (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, United States). DAPI was used to detect nuclei in a fixed and permeabilized HLE-B3 cells. All images of immunostained HLE-B3 cells, were taken with a Nikon C1Plus confocal microscope using NIS-Elements acquisition software (Nikon, Tokyo, Japan).
6
Immunofluorescence Staining of HDAC1 and GCN5
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For immunofluorescence studies of HDAC1 and GCN5, cells were treated with the psychostimulants and opioid with or without piracetam and grown in chamber slides. For immunostaining cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min at room temperature and then blocked with 5% normal goat serum at 4°C for 1h and then incubated with antibodies: anti-HDAC1 (Cell Signaling Technology) and anti-GCN5 (Proteintech) overnight at 4°C. After washing with 1X PBS, cells were incubated with anti-mouse-IgG Alexa Fluor® 633 for GCN5 and anti-rabbit-IgG Alexa Fluor® 546 for HDAC1 for 2h. The cellular nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI). Slides were examined with a Nikon C1plus confocal microscope.
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