A549 cells were grown on glass coverslips and infected with the indicated viruses at an MOI of 1 FFU (fluorescence-forming unit)/cell. At the indicated time points post infection, cells were fixed for 15 min in 2.5% formaldehyde, permeabilized in 0.2% Triton X-100, and stained with specific antibody against NP antigen (clone AA5H, Bio-Rad/Serotec, 1:1000, #MCA400) in 3% BSA for 1 h. After washing, cells were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:1000) in 3% BSA. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) by adding to the secondary antibody solution. Coverslips were mounted using Mowiol and images were acquired by an Eclipse A1 laser-scanning microscope using the NIS-Elements software package (Nikon). At least 140 cells were counted per condition to quantify the subcellular distribution of NP using the ImageJ software.
Eclipse a1
Manufactured by Nikon
Sourced in Japan
The Eclipse A1 is a compact, automated microscope system designed for routine observation and imaging of biological samples. It features a high-quality optical system, including a high-intensity LED illumination source and a sensitive CCD camera, to provide clear, detailed images. The Eclipse A1 is suitable for a variety of applications in life science research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 555, by Thermo Fisher Scientific (2 mentions)
Leitz dm6000b microscope, by Leica (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Nis elements software, by Nikon (1 mentions)
Vectashield h 1000, by Vector Laboratories (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
7 protocols using eclipse a1
1
Quantifying Viral Nucleoprotein Localization
2
Cell Cycle Exit Analysis in Developing Cortex
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For the 20 h EdU pulse-chase assay, time-pregnant females were injected intraperitoneally with 50 mg/kg bodyweight EdU. After 20 h, females were killed, and embryo tissue processed as described above. Slides were incubated in 2 N HCl at 37°C for 20 min and rinsed two times in PBS with 0.2% Triton X-100. EdU detection was performed after immunostaining according to manufactures Click-it EdU Alexa 594 imaging kit protocol (Life Technologies). The fraction of cells that had exited the cell cycle (Q-fraction) was estimated by counting the numbers of EdU+/Ki67− cells basal to the subventricular zone (SVZ) and all EdU+ cells in 200 μm cortical segments. Q-fractions were calculated by dividing the number of EdU+/Ki67− cells by the total number of EdU+ cells. All imaging was carried out on a Nikon Eclipse A1 laser scanning confocal microscope. Merge-channel views were acquired with the associated NIS Elements A1 software.
3
S1PR4 Internalization and Recycling Assay
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Stable C6 glioma cells expressing EGFP-conjugated S1PR4 were prepared by infection with retrovirus bearing S1PR4–EGFP fusion construct (kindly provided by Dr Jerold Chun at Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA). S1PR4 internalization and recycling were assessed as previously described.44 (link) In brief, cells were plated on poly-L-lysine (100 μg/mL)-coated coverslips, cultivated, serum-deprived, and then used for experiments. The cells were treated with vehicle (0.1% fatty acid-free bovine serum albumin), S1P, FTY720-P, or SLB736 for 0.5 hours; in some cases, the cells were washed and further incubated in the presence of cycloheximide (5 μg/mL) for 2 or 4 hours. At the end of each experiment, the cells were fixed in 4% paraformaldehyde and mounted with Vectashield (H-1000; Vector Laboratories, Burlingame, CA). S1PR4 localization in cells was assessed by detecting the EGFP signal using laser scanning confocal microscopy (Eclipse A1+; Nikon, Tokyo, Japan).
4
Immunofluorescence Analysis of Photoreceptor Proteins
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Images were obtained using a Nikon Eclipse A1 scanning confocal microscope, controlled and processed using Nikon Elements software. For high magnification and high resolution immunofluorescence analysis eyes of C57Bl/6J mice were cryofixed, sectioned, and immunostained as previously described38 (link),39 (link) for two independent experiments. Cryosections were double stained for PRPF6, PRPF8 (A5463), GPR20, MAS1, SREB3, HTR1B (1:200) and centrin-3 (1:100) as a molecular marker for the connecting cilium, the basal body, and the adjacent centriole of photoreceptor cells40 (link). Subsequently, cryosections were incubated with the according secondary antibodies conjugated to Alexa 488 or Alexa 555 (LifeTechnologies), counterstained with DAPI (Sigma-Aldrich), and mounted in Mowiol 4-88 (Hoechst). Specimens were analysed and deconvoluted in a Leica LEITZ DM6000B microscope (Leica). Images were processed with Adobe Photoshop CS using different tools including contrast and colour correction as well as pixel extrapolation.
5
Immunofluorescence Analysis of Photoreceptor Proteins
6
Immunofluorescence Staining of Dissociated Cardiac Cells
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The dissociated samples or purified AMs were seeded on vitronectin-coated glass bottom plates or coverslips and fixed with 2% paraformaldehyde for 20 min at room temperature (RT), followed by permeabilization with 0.1% Triton X100 for 8 min at RT. After blocking for 1 h at RT with 5% fetal bovine serum (FBS), the cells were incubated at 4 °C overnight with the primary antibodies COUP-TFI, COUP-TFII, cTnI, or NKX2.5, as indicated in Supplementary Materials section F . After washing, the cells were incubated for 1 h at RT with secondary antibodies AF488 or Cy3 and 5 min at RT with DAPI, and the coverslips were mounted with ProLong-Gold. Confocal images were captured using a Leica SP5 or Nikon Eclipse A1. See Supplementary Materials F for more information.
7
Confocal Microscopy of Solid Food
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2.2.2.2.1 Confocal laser scanning microscopy of solid food before and after freeze-drying Sections of 0.1-mm thickness were cut with a sharp razor blade and placed on microscopy slides between two gene frames with 10 µL of fluorescent probes for protein (Alexa Fluor 546, Thermo
Fisher Scientific, Inc., USA) and fat (Nil Red, Thermo Fisher Scientific, Inc., USA). Slides were examined using a confocal microscope (green laser) (Eclipse A1+, Nikon, Japan) in spectral mode.
Pictures taken (presented here) were representative of the whole slide.
Fisher Scientific, Inc., USA) and fat (Nil Red, Thermo Fisher Scientific, Inc., USA). Slides were examined using a confocal microscope (green laser) (Eclipse A1+, Nikon, Japan) in spectral mode.
Pictures taken (presented here) were representative of the whole slide.
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