To evaluate and compare apoptotic cell death in drebrin cKO and littermate control mouse lenses, embryonic E18.5 and P1 stage tissue cryosections were subjected to in-situ Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) using an ApopTag Plus Fluorescein Kit (EMD Millipore, Burlington, MA, USA) as we described earlier.12 (link) TUNEL labeled tissue sections were counter-stained with propidium iodide. Imaging was performed using a Nikon Eclipse 90i confocal microscope in conjunction with manual counting of TUNEL positive cells.
Eclipse 90i confocal microscope
Manufactured by Nikon
Sourced in United States
The Eclipse 90i confocal microscope is a high-performance laboratory instrument designed for advanced imaging applications. It features a modular design and supports a range of imaging techniques, including confocal, wide-field, and multi-photon microscopy. The Eclipse 90i is equipped with a powerful laser system, precise motorized stages, and state-of-the-art optical components to provide researchers with detailed, high-resolution images of samples.
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Lab products found in correlation
Cyanine3 (cy3), by Nikon (3 mentions)
Fluoromount g, by Electron Microscopy Sciences (2 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (2 mentions)
Eub338 16s rrna gene probe, by Thermo Fisher Scientific (2 mentions)
D223 28s rrna gene probe, by Thermo Fisher Scientific (2 mentions)
5 cy3 fluorophore, by Thermo Fisher Scientific (2 mentions)
Polyclonal antibody to sap, by Biocare Medical (2 mentions)
Apoptag plus fluorescein kit, by Merck Group (1 mentions)
Image it fx signal enhancer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole counterstain, by Serva Electrophoresis (1 mentions)
Ax70 fluorescent microscope, by Nikon (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugate of wheat germ agglutinin, by Thermo Fisher Scientific (1 mentions)
Ix81 inverted fluorescence microscope, by Olympus (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Shandon cytospin 2, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
20 protocols using eclipse 90i confocal microscope
1
Apoptosis Analysis in Mouse Lenses
2
Immunofluorescence Microscopy of PCa Cells
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PCa cells were seeded on glass coverslips and were subsequently treated with the different agents. Cells were fixed with 4% paraformaldehyde in PBS. For blocking background staining from nonspecific interactions, Image‐iT™ FX signal enhancer (Molecular Probes, Inc, Eugene, OR) was used. The secondary antibodies including rabbit antidonkey conjugated to Rhodamine or Alexa Fluor 555 (Chemicon/Millipore International Inc, Temecula, CA) or antigoat conjugated to FITC antibodies and goat antirabbit Alexa Fluor 488 (Invitrogen, Stockholm, Sweden) were used. 4′,6‐Diamidino‐2‐phenylindole counterstain (SERVA Electrophoresis GmbH, Heidelberg, Germany) was used to visualize cell nuclei. The images were viewed and taken under an Olympus AX70 fluorescent microscope or a Nikon Eclipse 90i Confocal microscope (Nikon DS‐U1) and software ACT2U (ACT2U version. 1.5, Stockholm, Sweden) was used.
3
Confocal Microscopy Quantification Protocol
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Confocal images were acquired using a Nikon Eclipse 90i confocal microscope with 488-nm excitation and 515-nm emission filters for samples probed with the FITC conjugated peptide. Images of samples stained with Thioflavin T or calcofluor white were acquired with 408-nM excitation and 450-nM emission filter settings. Quantified images were analyzed using the Nikon EZ-C1 Gold version 3.90 software. Fluorescent images, representing a single field per sample, were opened and set to view in 1D graph mode with the x-axis set. The highest fluorescent peak expressed in arbitrary units was noted for four y-axis positions in each field. The mean and standard deviation were determined for the four y-axis positions in four fields.
4
Immunofluorescence Imaging of Transfected Cells
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Cells were grown on glass coverslips in 24‐well plates, transfected as described. Cells were fixed stained and mounted as previously described (Thirkettle et al., 2009a (link)). Images were taken using a Nikon eclipse 90i confocal microscope with 60× objective. All scale bars represent 10 μM.
5
Retinal Imaging and Photoreceptor Quantification
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Agarose-embedded retinal cross sections were prepared as previously described (Lobanova et al., 2010 (link)), collected in 24-well plates, and incubated for 2 h with Alexa Fluor 594 conjugate of wheat germ agglutinin (Invitrogen) in PBS containing 0.1% Triton X-100. Sections were washed three times in PBS, mounted with Fluoromount G (Electron Microscopy Sciences) under glass coverslips, and visualized using a Nikon Eclipse 90i Confocal Microscope.
Plastic-embedded retinal cross sections (1 μm thick) were prepared as previously described (Sokolov et al., 2004 (link)) and stained with toluidine blue for light microscopy. Tiled images of whole retina cross sections were obtained using the Olympus IX-81 Inverted Fluorescence Microscope, and aligned and stitched using the Olympus cellSens Dimension software. The number of photoreceptor nuclei in representative segments of outer nuclear layer (ONL) was quantified as a quantitative measure of surviving photoreceptors. The number of nuclei in a 400 μm segment of the ONL, located at 1 mm from each side of the optic nerve, was counted by hand.
Plastic-embedded retinal cross sections (1 μm thick) were prepared as previously described (Sokolov et al., 2004 (link)) and stained with toluidine blue for light microscopy. Tiled images of whole retina cross sections were obtained using the Olympus IX-81 Inverted Fluorescence Microscope, and aligned and stitched using the Olympus cellSens Dimension software. The number of photoreceptor nuclei in representative segments of outer nuclear layer (ONL) was quantified as a quantitative measure of surviving photoreceptors. The number of nuclei in a 400 μm segment of the ONL, located at 1 mm from each side of the optic nerve, was counted by hand.
6
Optimizing Cytokinesis Synchronization Imaging
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For immunostaining or Western blotting, the following primary antibodies were used: anti-phospho-Histone H3 (Ser-10) (32219, Upstate), Histone H3 (9715 S, Cell Signaling), α-Tubulin (3873 S, Cell Signaling), Ki-67 (15580, Abcam) and RhoA (179, Santa Cruz). A Nikon Eclipse 90i Confocal Microscope coupled with the EZ-C1 Imaging Software was used for imaging. For cytokinesis synchronization optimization, at least 10 shots were taken from different spots per slide in 60x magnification and images were quantified using the ImageJ [Wayne Rasband, NIH] software.
7
Immunostaining of Drosophila Testes
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Adult male flies were placed in ethanol for 2 to 5 min before dissection. Testes were dissected in phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). Samples were washed and blocked in a solution of 0.5% bovine serum albumin in 0.3% PBST (PBS containing 0.3% Triton X-100) for 1 hour. Testes were incubated in primary antibody in blocking solution overnight at 4°C. After washing, a secondary antibody was added in the blocking solution and incubated at RT for 1 hour, washed, and placed at 4°C in 70% glycerol. Samples were mounted in Vectashield (Vector Laboratories). 4′,6-Diamidino-2-phenylindole (DAPI) staining was performed using DAPI in Vectashield. Rhodamine-phalloidin (1:200) was used to visualize filamentous actin. Images were viewed on a Nikon Eclipse 90i confocal microscope with EZ-CI 3.80 software. Antibodies are listed in Supplementary Materials and Methods in the Key Resources Table.
Larval testes were fixed and stained in four-well plates with mesh baskets and directly mounted in Vectashield. For immunostaining of Bam in larval testes, incubation in primary antibody was performed for three nights at 4°C. Bam antibody (1:50) was added each night, and the staining protocol was resumed on day 4.
Larval testes were fixed and stained in four-well plates with mesh baskets and directly mounted in Vectashield. For immunostaining of Bam in larval testes, incubation in primary antibody was performed for three nights at 4°C. Bam antibody (1:50) was added each night, and the staining protocol was resumed on day 4.
8
Immunocytochemical Detection of Insulin and EMCV
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Dispersed islet cells were washed twice in PBS, resuspended in PBS, and centrifuged onto microscope slides using a Shandon Cytospin II (ThermoFisher Scientific). Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min and blocked using 1% BSA in PBS with 0.2% Tween (PBST). Primary antibodies to insulin and EMCV capsid were used at 1:1000 in PBST for 1 h. Secondary antibodies Cy3-conjugated donkey anti-guinea pig and AF-488-conjugated donkey anti-rabbit were used at 1:1000 in PBST in a dark, humidified chamber for 1 h. ProLongTM Gold Antifade Reagent with DAPI (Invitrogen) was used to preserve fluorescent signal and for nuclear staining. Images were captured using a Nikon eclipse 90i confocal microscope.
9
Quantitative Fluorescence Microscopy for RPE
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Fluorescence microscopy was performed using Nikon Eclipse 90i confocal microscope equipped with ×20 air objective. For the quantification, maximum intensity projection images were extracted from each z-stack using Fiji, an open-source image processing software 36. The number of RPE cells was counted using Fiji software within the corresponding area.
10
Retinal Morphology Evaluation via Microscopy
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Retinal morphology was evaluated in semi-thin plastic-embedded retinal cross-sections (0.5 μm thick) obtained from mice after a/LCI and OCT imaging. Sections were prepared as described in Lobanova et al.43 (link),44 (link) and stained with toluidine blue for light microscopy. For immunohistochemistry, agarose-embedded retinal cross-sections were prepared and collected in 24-well plates45 (link), and incubated overnight with biotinylated anti-β-amyloid, primary antibody (6E10; Covance # SIG-39340), followed by 2 h incubation with DyLight streptavidin 488 secondary antibody (Vector laboratories #SA-5488) in PBS containing 0.1% Triton X-100. Sections were washed three times in PBS, mounted with Fluoromount G (Electron Microscopy Sciences) under glass coverslips, and visualized using a Nikon Eclipse 90i confocal microscope.
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