HeLa cells were cultured on glass coverslips at a density of 105 cells per cm2. After 16 h cells were transfected with 300 ng of expression plasmids using the Lipofectamine 2000 transfection reagent. 24 h after transfection cells were rinsed with PBS, fixed with 4% paraformaldehyde (PFA, Merck) in PBS (pH = 7.5). Specimens were embedded in “ProLong Gold antifade” reagent (Thermofisher) supplemented with 2-(4-carbamimidoylphenyl)-1H-indol-6-carboximidamide (DAPI) and stored at 4°C overnight. Pictures were taken using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu), and a 100-W mercury lamp (HBO 103W/2, Osram). The following filter sets were used: Green, (EGFP) ex: HQ470/40, em: HQ525/50, blue (DAPI) D360/50, em: D460/50.
C4742
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C4742 is a high-performance CCD camera system designed for scientific and industrial applications. It features a high-resolution CCD sensor, advanced image processing capabilities, and a user-friendly interface. The C4742 is suitable for a wide range of applications, including microscopy, spectroscopy, and imaging applications. For a more detailed and unbiased description, please consult the product's technical documentation.
Automatically generated - may contain errors
Lab products found in correlation
Hbo 103w 2, by Osram (7 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Chamber coverslips, by Thermo Fisher Scientific (2 mentions)
Application suite software, by Leica (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Iplab, by BD (1 mentions)
Lsm510 laser scanning confocal microscope, by Zeiss (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Chambered coverslips, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
12 protocols using c4742
1
Fluorescence Imaging of Transfected HeLa Cells
2
Immunofluorescence Staining of Pancreatic Sections
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H&E and immunofluorescence staining of frozen pancreatic sections was performed as described previously.8 (link), 18 (link) For immunofluorescence staining, the following antibodies were used: polyclonal guinea pig anti-insulin serum (cat. no. A0564; Dako, Carpinteria, CA, USA), rat anti-CD8 (cat. no. MCA2694; AbD Serotec, Oxford, UK), or rat anti-CD4 (cat. no. MCA1767GA; AbD Serotec), anti-guinea pig IgG-fluorescein isothiocyanate (FITC) (cat. no. F-6261; Sigma-Aldrich, St. Louis, MO, USA) and anti-rat IgG-TRITC (cat. no. T4280; Sigma-Aldrich). Sections were covered with Cytoseal60 mounting medium (cat. no. 18006, Electron Microscopy Sciences, Hatfield, PA, USA). Images were captured with an Olympus IX71 fluorescence microscope equipped with a digital camera (C4742, Hamamatsu). Editing of the pictures was performed using ImageJ software (https://imagej.nih.gov/if/ ). Images of H&E-stained sections were acquired on a light microscope (Leica, Germany) equipped with a digital camera and Leica Application Suite software (Leica Microsystems, Switzerland).
3
Fluorescence Microscopy of ISC1-GFP
4
Fluorescent Microscopy of Transfected HeLa Cells
5
Imaging GFP-fusion Proteins in HeLa Cells
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HeLa cells were plated (1 × 105 cells/cm2) on chamber coverslips (Nunc). After 18 h, cells were transfected with 150 ng of expression plasmids for the specific GFP-fusion proteins. After 24 h, cells were rinsed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Specimens were embedded in ProLong© Gold antifade reagent (Invitrogen) supplemented with [2-(4-carbamimidoylphenyl)-1H-indol-6-carboximidamide] (DAPI) and stored at 4 °C overnight. Cells were imaged using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu) and a 100-W mercury lamp (HBO 103 W/2, Osram) using the following filter sets: GFP detection, ex: HQ470/40, em: HQ525/50, DAPI detection, ex: HQ360/40, em: HQ457/50.
6
Visualizing NICD1 Localization in HeLa Cells
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Imaging was performed by plating HeLa cells at a concentration of 5 × 105 cells/cm2 on chamber coverslips (Nunc). After 16 h, cells were transfected with 250 ng of expression plasmids GFP-NICD1 wt or GFP-NICD1 8KR using the Nanofectin transfection reagent (PAA). Cells were fixed (4% paraformaldehyde) 24 hours after transfection. After DAPI (4′,6-diamidino-2-phenylindole) staining, pictures were acquired using a IX71 fluorescence microscope (Olympus) equipped with a digital camera (C4742, Hamamatsu) and a 100-W mercury lamp (HBO 103W/2, Osram). The following filter sets were used: green channel (ex: HQ470/40, em: HQ525/50), blue channel: (ex: HQ360/40, em: HQ457/50).
7
Quantification of Postsynaptic Endplate Morphology
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For quantification of postsynaptic endplate morphology, cultures were digitally photographed using a wide-field fluorescence microscope equipped with a broad focal plane lens (Leica Microsystems, Bannockburn, IL, USA) attached to a digital camera (C4742; Hamamatsu, Japan). Synapses were only quantified if the captured imaged accurately reflected the entire three-dimensional structure of the synapse. Captured images were analyzed for areas using IPLab (Version 4.0; BD Biosciences) or ImageJ software. All representative images are shown as collapsed z-stacks acquired using an LSM510 laser scanning confocal microscope (Zeiss Microimaging, Thornwood, NY, USA) and managed using Zen 2009 software (Zeiss Microimaging).
8
Fluorescence Imaging of Protein Localization
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Cell imaging was performed by plating HeLa cells or HEK293 cells in a concentration of 105 cells cm−2 on chambered coverslips (Nunc). When required, cells were transfected with 400 ng of expression plasmids using the Nanofectin transfection reagent (see above). Cells were rinsed with PBS 24 h after transfection, fixed and permeabilized with 0.1% Triton X-100. Nonspecific immunostaining was blocked by incubating the cells in 3% BSA in PBS with 0.1% TWEEN-20. The following antibodies were used: anti-Flag, mouse monoclonal IgG (M5, F4042, Sigma), secondary antibody, Alexa-Flour-488 coupled goat anti-mouse IgG (A11011, Life Technologies), anti-SHARP.2 [rabbit polyclonal, (18 (link))], secondary antibody, Alexa-Fluor-568 coupled goat anti-rabbit IgG, (A11011, Life Technologies), anti-MLL2/KMT2D, goat polyclonal (I18, sc68671, Santa Cruz), secondary antibody, Alexa-Fluor-488 coupled donkey anti-goat (A11055, Life Technologies). Pictures were taken using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu), and a 100-W mercury lamp (HBO 103W/2, Osram). The following filter set was used: Green, (Alexa-Flour-488) ex: HQ470/40, em: HQ525/50. For confocal microscopy a Leica TCS SP8-HCS microscope was used. Fluorophores were excited with the 488 nm and the 561 nm laser lines, respectively. Pictures were taken as ARY-sections in sequential scan mode.
10
GFP-RBPJL Transfection in HeLa Cells
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HeLa cells were plated (1 × 105 cells/cm2) on chamber coverslips (Nunc-Lab-Tek, #155380). After 18 h, cells were transfected with 150 ng of GFP-RBPJL specific expression plasmids. 24 h after transfection, living cells were imaged using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu) and a 100-W mercury lamp (HBO 103W/2, Osram). Filter set for GFP detection: excitation, HQ470/40; emission, HQ525/50. Filter set for DAPI detection: D360/50; emission: D460/50.
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