Vero cells were grown in glass coverslips and non-infected or infected with rBTV-1, rBTV-4, rBTV-8, rBTV-1/Venus, rBTV-4/Venus, or rBTV-8/Venus at a MOI of 0.1. Twenty-four hours after infection, cell monolayers were fixed for 15 min with paraformaldehyde 4%. Fixed cells were blocked with 20% FBS-PBS-0.2% saponine (20% blocking solution) for 60 min at room temperature (RT). Cells were then incubated overnight at 4°C with a mouse hyperimmune serum against NS1 (generated in the laboratory) (61 (link)) (1:500) or the NS2 BTV-specific monoclonal antibody (mAb) 23H6 (Eurofins INGENASA, Madrid, Spain) (1:500) diluted in 20% blocking solution. After three serial washing steps with PBS, cells were incubated for 30 min at RT with Alexa Fluor 594 goat conjugated anti-mouse IgG (Invitrogen, German Town, MD, USA) (1:1,000). Coverslips with infected Vero cells were washed three times with PBS and once with PBS-DAPI (1:10,000). Laser scanning confocal microscopy images were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analyzed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages.
Axiovert lsm 880 microscope
Manufactured by Zeiss
The Axiovert LSM 880 microscope is a high-performance laser scanning confocal microscope designed for advanced imaging applications. It features a flexible and modular design, allowing for customization to meet specific research requirements. The Axiovert LSM 880 is capable of capturing high-resolution, three-dimensional images with exceptional clarity and detail.
Automatically generated - may contain errors
4 protocols using axiovert lsm 880 microscope
1
Visualization of BTV Nonstructural Proteins
2
Immunofluorescent Staining of Trout IgM+ B Cells
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FACS isolated HK IgM+/CD38- and IgM+/CD38+ B cell populations were collected as described above and seeded on a poly-L-lysine (0.01% solution, Sigma)-coated slides and incubated at RT for 30 min in a humidified chamber. The slides were then fixed in 4% paraformaldehyde solution for 30 min at RT. The fixed samples were incubated for 1 h at RT with a blocking solution (TBS, pH 7.5 containing 5% BSA and 0.5% saponin) to permeabilize the cells and to minimize non-specific adsorption of the antibodies to the coverslip. The samples were then incubated with a mAb against trout IgM coupled to APC (17 mg/ml) for 1 h at RT in a humidified chamber. Slides were counterstained with 1 μg/ml DAPI (Sigma-Aldrich) for 10 min at RT, rinsed with PBS 1x and mounted with Fluoromount (Sigma-Aldrich) for microscopy. Laser scanning confocal microscopy images were acquired with an inverted Zeiss Axiovert LSM 880 microscope with Zeiss Zen software. Images were analyzed and processed with Zeiss Zen and Adobe Photoshop CS6 software packages.
3
Visualizing APRIL Binding to Trout IgM+ B Cells
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To further investigate the capacity of APRIL to bind trout IgM+ B cells, splenocytes were incubated with 1 µg/ml of recombinant biotinylated APRIL in L-15 media supplemented with 5% FCS. After 15 min at 20°C, the cells were washed with serum-free L-15 medium, seeded on poly l -lysine coated slides, and incubated at 20°C for 30 min. After gently washing with PBS, the slides were fixed in 4% PFA for 15 min at RT and then incubated for 1 h at RT with blocking solution (PBS, pH = 7.5, containing 0.01% BSA, 0.02% Tween-20, and 0.5% saponin). Fixed cell slides were then incubated with APC-anti-IgM mAb (1 µg/ml) and FITC-streptavidin (0.5 µg/ml) for 1 h. Samples were counterstained with 1 µg/ml DAPI (Sigma). Laser scanning confocal microscopy images (0.3-µm thickness) were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analyzed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages.
4
Immunofluorescent Staining of Trout Gill Cells
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Rainbow trout gills from anesthetized and exsanguinated rainbow trout were embedded in PolyFreeze cryostat mounting medium (Sigma), immediately frozen in liquid nitrogen, and stored at −80○C until used. Cryostat sections with a thickness of 14 µm were prepared using a Leica CM3050 microtome and placed on SuperFrost glass slides (Menzel-Gläser). Dry sections were fixed in acetone at −20°C for 20 min, air dried, encircled with a hydrophobic compound (ImmunoPen; Calbiochem), incubated for 1 h at room temperature with a blocking solution (TBS buffer pH 7.5, containing 0.01% BSA, 0.5% saponin, 0.02% Tween-20, and 5% goat serum), and stained with Abs against trout MHC II β-chain (allophycocyanin-labeled) and trout CD8α. Samples were washed and incubated with Alexa Fluor 488 F(ab′)2 fragment of goat anti-rat igG (H + L) (Life Technologies). Samples were counterstained with 1 µg/ml DAPI (Sigma). Laser scanning confocal microscopy three-dimensional image stacks (12 µm total thickness) were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analyzed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages.
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