For analysis of binding of GFP- or mCherry-tagged nanobodies to the cell surface of C. reinhardtii and other algae using confocal microscopy, cells in 0.5 mL of cell culture at saturation density were collected by centrifugation at 5000 × g for 2 minutes in a 1.5 mL centrifuge tube. Cell pellets were re-suspended in 0.5 mL TAP medium containing 1% non-fat dry milk and then shaken for 15 min in light. Cells were washed twice with TAP medium and re-suspended in 0.5 mL TAP medium containing 1% non-fat dry milk, followed by the addition of chimeric mCherry or GFP VHH B11 nanobody to the desired final concentration, typically 30 nM. Fluorescence BoNT VHH B5 served as negative control. After shaking for 30 min in light, cells were washed twice with TAP medium. Cells were then examined by confocal fluorescence microscopy using a Nikon ECLIPSE 90i system at 1000× magnification. The excitation wavelength was set at 561.5 nm and the emission wavelength at 570-620 nm for mCherry fluorescence, 448 nm and 500–550 nm for GFP fluorescence and at 641 nm and 662-737 nm for chlorophyll auto-fluorescence to ensure no cross talk between different fluorescence channels.
Eclipse 90i system
Manufactured by Nikon
Sourced in Japan
The Eclipse 90i system is a high-performance microscope designed for advanced imaging and analysis applications. It features a modular design that allows for customization to meet specific research or industrial requirements. The core function of the Eclipse 90i is to provide a versatile and reliable platform for microscopic observation and data collection.
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Lab products found in correlation
6 protocols using eclipse 90i system
1
Nanobody Binding Analysis in Algae
2
Histological Analysis of Osteoclastogenesis
3
Multicolor 3D-FISH Microscopy Imaging
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Metaphase FISH images were captured with a Nikon Eclipse 90i system, equipped with Nomarski differential interference contrast (DIC) optics. Samples were photographed with a DS-5Mc Nikon digital camera and the resulting photographs were analyzed by using the Nikon AcU2 software program. Light optical serial sections of nuclei studied in three color 3D-FISH experiments were obtained with a laser scanning confocal microscope (LSM 410; Carl Zeiss MicroImaging) equipped with Ar and He/Ne lasers. For imaging of multicolor 3D-FISH experiments a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems) with beam splitters tuned for DAPI, Alexa 488, and TexasRed was used. Nuclei were scanned with an axial distance of 200 nm between consecutive light optical sections yielding separate stacks of 8-bit gray-scale images for each fluorescence channel with a pixel size of 60 nm. For each optical section, images were collected sequentially for all fluorochromes used, followed by correction of the axial chromatic shift for each fluorescence channel as described by Walter et al.23 (link) Confocal image stacks were processed with ImageJ software (http://rsb.info.nih.gov/ij ) using the deconvolution plugin to enhance resolution.
4
Histological Analysis of Calvarial Osteoclastogenesis
5
Immunohistochemical Analysis of DDX20 in Testis
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The immunohistochemical (IHC) staining of the paraffin-embedded testicular samples of the local cohort was implemented to investigate the protein expression levels and distribution of DDX20. The testicular slides were de-paraffinized in xylene and then added to the ethanol following the below concentration: 100% ethanol (4 min), 90% ethanol (4 min), 80% ethanol (4 min), and 70% ethanol (4 min). Subsequently, the slides were blocked in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with the primary antibody (rabbit anti-DDX20, 1:100, Proteintech, China) at 4°C overnight. After washing the slides 3 times with PBS, we incubated the slices with anti-rabbit secondary antibodies (Proteintech, China). Nikon Eclipse 90i system (Nikon, Japan) was used to get the images, and the Image-Pro Plus (version 6.0, Media Cybernetics, USA) software was used to measure the integral optical density (IOD), which represented the protein expression levels. For each sample, 3 slices were randomly chosen to conduct the IHC staining, and 5 different microscopic fields of a slide were randomly selected to evaluate the levels of DDX20.
6
Histological Assessment of Inflammation and Osteoclastogenesis in Mice
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Mice were sacrificed on day 7, and the calvaria and adjacent connective tissues were dissected for histological assessment of inflammation and osteoclastogenic activity. Specifically, the tissues were fixed in 4% phosphate‐buffered formalin and then decalcified in 10% EDTA. The parietal bones were bisected into rostral and caudal sides and embedded in paraffin. Five nonconsecutive coronal sections of 5 µm thickness were prepared. Sections were stained with hematoxylin and eosin (HE) or with a leukocyte acid phosphatase kit (Sigma‐Aldrich) for tartrate‐resistant acid phosphatase (TRAP) activity. TRAP‐positive multinucleated cells (MNC, ≥3 nuclei) were counted as mature osteoclast. Images were acquired using a Nikon Eclipse 90i system (Nikon). Quantification of osteoclasts was performed on five randomly selected coronal sections/mouse and expressed as mean cell numbers per section. TRAP staining of the whole calvarial bone without soft tissues was also done to assess the overall osteoclastogenic activity on calvaria after Pg infection.
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