After blocking with 10% goat serum (Vector Laboratories), spinal cord sections were incubated with antibodies for APC (1:100, clone CC1, Abcam), Ki67 (1:200, clone SoIA15, eBioscience), and Olig2 (1:200; cat. no. AB9610, Millipore) overnight. Secondary antibodies goat anti-rabbit AF488 (1:200; cat. no. A-11008, Life Technologies) and goat anti-rat AF594 (1:200; cat. no. A-11007, Life Technologies) were incubated for 1 hr at RT. For CC1 detection acidic antigen retrieval was performed and a mouse-on-mouse immunodetection kit was used (Vector Laboratories). Nuclei were counterstained with DAPI before coverslipping with ProLong Gold Antifade (Life Technologies). Spinal cord images were acquired on a Leica DM5500 epifluorescence microscope.
Dm5500 epifluorescence microscope
Manufactured by Leica
The Leica DM5500 is an epifluorescence microscope designed for advanced microscopy applications. It features high-intensity illumination and a modular design to accommodate a variety of fluorescence and contrast techniques.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum, by Vector Laboratories (2 mentions)
Clone cc1, by Abcam (2 mentions)
Clone soia15, by Thermo Fisher Scientific (2 mentions)
Goat anti rat af594, by Thermo Fisher Scientific (2 mentions)
Mouse on mouse immunodetection kit, by Vector Laboratories (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Aniline blue, by Merck Group (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
10 protocols using dm5500 epifluorescence microscope
1
Immunohistochemical Analysis of Spinal Cord
2
Quantifying Oocyte and Sperm Development
3
Quantifying Callose Deposition in Maize Leaves After Aphid Feeding
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Ten adult aphids were confined to a clip cage for 72h on the third leaf of two-week-old maize seedlings. Control plants received cages without aphids. Three days later, leaf material within the aphid cage was excised and used for callose staining as described previously (Luna et al., 2011 (link); Ton et al., 2005 (link)). Leaves were de-stained in 98% ethanol for at least 48h until all tissue was transparent, washed in 0.07M phosphate buffer (pH 9), incubated (stained) for 2h in 0.07M pH 9 phosphate buffer containing 0.01% aniline-blue (Sigma, St. Louis, MO), and stored at 4 °C in 0.07M pH 9 phosphate buffer until microscopic analysis. Observations were performed with an Leica DM5500 epifluorescence microscope equipped with a ×20 immersion objective, a UV filter (BP 340 to 380nm, LP 425nm) a Retiga-2000R colour CCD camera, and Qcapture Pro 6.0 acquisition software (Leica, Wetzlar, Germany). Callose spots were quantified on the adaxial side of the leaf segment contained within the clip cage (~146mm2, with or without aphid feeding) and number of callose spots was calculated per mm2 of leaf tissue.
4
Immunohistochemical Analysis of Spinal Cord
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After blocking with 10% goat serum (Vector Laboratories), spinal cord sections were incubated with antibodies for APC (1:100, clone CC1, Abcam), Ki67 (1:200, clone SoIA15, eBioscience), and Olig2 (1:200; cat. no. AB9610, Millipore) overnight. Secondary antibodies goat anti-rabbit AF488 (1:200; cat. no. A-11008, Life Technologies) and goat anti-rat AF594 (1:200; cat. no. A-11007, Life Technologies) were incubated for 1 hr at RT. For CC1 detection acidic antigen retrieval was performed and a mouse-on-mouse immunodetection kit was used (Vector Laboratories). Nuclei were counterstained with DAPI before coverslipping with ProLong Gold Antifade (Life Technologies). Spinal cord images were acquired on a Leica DM5500 epifluorescence microscope.
5
Quantifying Cell Death in VNO
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Caspase 3 fluorescent IHC staining on VNO cryosections was imaged with a Leica DM5500 epifluorescence microscope, with the 63× objective, and images centered on single transgene-expressing cells were acquired. For quantification, we defined a 50-μm radius around the transgene-expressing cell and counted all cells within this area based on the DAPI staining. The cell death rate was estimated from the number of Caspase 3–labeled cells over the total number of either transgene-expressing cells or neighboring cells.
6
Quantification of Alzheimer's Pathology
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Brain sections for counting of Aβ plaques, and reactive astrocytes and microglia, were imaged using a Leica SP8 confocal laser scanning microscope, equipped with a HC PL APO 20x/NA0.75 objective (Leica, #15506517). A Leica DM5500 epifluorescence microscope with a HC PL APO 10x/NA0.45 objective (Leica, #506411) was used to take the mosaic image in Fig 2C and the single fields of view in Fig EV3C–F . Quantification of Aβ plaque number and reactive astro‐ and microgliosis was carried out in a semi‐automatized fashion with the aid of ImageJ/Fiji software.
7
Fluorescent Nuclear Imaging of Plant Tissues
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Leaf and root tissue were harvested and fixed in a 3:1 ethanol:acetic acid solution. Fixed tissue was rehydrated in water and stained using the fluorescent dye DAPI at a concentration of 2 µg ml–1. Samples were de-stained by incubation in water, mounted on a slide plus coverslip, and imaged using a Leica DM5500 epifluorescence microscope. For guard cell nuclei, images were analyzed using FIJI (https://fiji.sc/ ) software to generate nuclear size and roundness index measurements.
8
Fpr Transgene Cell Quantification
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For the identification and the quantification of Fpr transgene-expressing cells, images were acquired with a Leica DM5500 epifluorescence microscope using the 20× objective centered around one VNO side. Cells were counted and labeled manually on the acquired images.
9
Staining Anther Filaments with DAPI
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Anther filaments of flowering plants were first fixed in a 3:1 ethanol: acetic acid solution for at least 20 min. Following a rinse in water, the filaments were briefly dipped in DAPI working solution (at 1 µg/ml) and then in water and placed on a slide for imaging using a Leica DM5500 epifluorescence microscope.
10
Leaf Tissue Staining and Microscopy
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Leaf tissue was boiled for 5 min in a 1:1 mixture of ethanol and staining solution (10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, and 10 mg of trypan blue, dissolved in 10 ml of distilled water) for staining. The leaf tissue was destained in chloral hydrate at 2.5 g/ml in distilled water. Microscopic analysis was done using a Leica DM5500 Epifluorescence Microscope.
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