NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described70 (link). Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).
Orca r2 charge coupled device camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The Orca-R2 is a charge-coupled-device (CCD) camera designed and manufactured by Hamamatsu Photonics. The camera features a high-resolution CCD sensor that captures images and data. The Orca-R2 is capable of precise and sensitive detection of light, making it suitable for a variety of scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Ctr 7000 hs controller, by Leica (5 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (4 mentions)
Dmi6000b microscope, by Leica (2 mentions)
Ix70 fluorescence microscope, by Olympus (1 mentions)
Axio observer, by Zeiss (1 mentions)
Axiocam 512 color camera, by Zeiss (1 mentions)
Discovery v8 microscope, by Zeiss (1 mentions)
Deltavision omx v4, by GE Healthcare (1 mentions)
Air therm atx, by World Precision Instruments (1 mentions)
Axiovision, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
12 protocols using orca r2 charge coupled device camera
1
Immunofluorescence Analysis of Salmonella Infection
2
Immunostaining of Nucleoflagellar Apparatus
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Nucleoflagellar apparatus prepared as previously described (Taillon and Jarvik, 1995 (link)) was fixed with 2% formaldehyde for 10 min at room temperature, followed by treatment with cold acetone and methanol (−20°C). Fixed samples were immmunostained as previously described (Sanders and Salisbury, 1995 (link)). Samples were observed by an IX70 fluorescence microscope (Olympus) with an ORCA-R2 charge-coupled device (CCD) camera (Hamamatsu Photonics, Shizuoka, Japan).
3
Immunofluorescence Analysis of Salmonella Infection
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NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described70 (link). Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).
4
Bacterial Growth and Imaging Protocol
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Overnight bacterial cultures were centrifuged (8,000 × g, 10 min, room temperature [RT]) and diluted 1:100 in LB at the desired pH (7.4, 5.8, or in the range 4.0 to 5.8) to exponential phase (optical density at 600 nm [OD600] ≈ 0.2 to 0.3). To maintain stable growth conditions (exponential phase), cultures were diluted 1:3 every 40 min in LB medium at the appropriate pH (5.8 or 7.4). After 2 h, bacteria were harvested (4,300 × g, 5 min, RT), washed in phosphate-buffered saline (PBS), and fixed with 3% paraformaldehyde. Images were acquired on an inverted Leica DMI 6000B microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).
5
Immunofluorescence Analysis of Salmonella Infection
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NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described70 (link). Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).
6
Bacterial Growth and Microscopy
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Bacteria in overnight cultures were centrifuged (4300 × g, 2 min, RT), washed in PCN medium with the corresponding pH (4.6 or 7.4) and salt concentration (0 mM or 200 mM NaCl), and used to inoculate the respective media to an initial optical OD600 of 0.02. After 4 h growing with agitation (150 rpm) at 37°C, bacteria were harvested (6800 × g, 4 min, 4°C), washed twice in PBS buffer, fixed with 3% paraformaldehyde (PFA) for 10 min, and adjusted to a final paraformaldehyde (PFA) concentration of 1%. For microscopy, fixed bacteria were centrifuged (4300 × g, 4 min, RT) and resuspended in an equal volume of PBS. A volume of 30 μl was dropped on poly‐L‐Lys precoated coverslips and incubated for 10 min at RT. Attached bacteria were washed four times with PBS and the coverslip was mounted on slides using ProLong Gold Antifade (Molecular Probes). Images were acquired on an inverted Leica DMI 6000B microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca‐R2 charge‐coupled‐device (CCD) camera (Hamamatsu Photonics).
7
Fluorescence Microscopy Imaging Protocol
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Fluorescence microscopy was carried out with a Zeiss Axio Observer.Z1 microscope (Carl Zeiss S.A.S.) with a × 63 oil immersion objective equipped with the following filter sets: FITC (Filter set 44, Excitation BP 475/40, Beam Splitter FT 500, Emission BP 530/50) for GFP, Propidium Iodide (Filter set 00, Excitation BP 530–585, Beam Splitter FT 600, Emission LP 615) for RFP. Cell contours were visualized with Nomarski optics. Images were acquired with an ORCA-R2 charge-coupled device camera (Hamamatsu). Images were treated and analysed with ImageJ.
8
Imaging Cultured Cells and Embryos
9
Mitochondrial Calcium Imaging with GCaMP6m
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To test resting mitochondrial Ca2+ concentrations with high sensitivity, we used a new Ca2+ probe based on the last-generation GCaMP probe (Chen et al., 2013 (link)) targeted to the mitochondrial matrix. We chose the GCaMP6m version because it had the highest Ca2+ affinity. To measure the signal independent of variations in basal fluorescence intensity due to the variable expression levels of the probe, we took advantage of the isosbestic point in the GCaMP6m excitation spectrum; exciting GCaMP6m at 406 nm led to fluorescence emission that was not Ca2+ dependent. As a consequence, the ratio between the excitation wavelengths of 494 and 406 nm was proportional to the Ca2+ concentration and independent of probe expression levels. Cells were imaged with an IX-81 automated epifluorescence microscope (Olympus) equipped with a 40× oil immersion objective (numerical aperture 1.35; Olympus) and an ORCA-R2 charge-coupled device camera (Hamamatsu Photonics).
10
Fluorescence Microscopy Imaging Protocol
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After treatment, cells were fixed by adding formaldehyde at a final concentration of 3,7% and incubated at room temperature with agitation for 30 mn. Cells were mounted between glass slide and coverslips after 2 subsequent washes with TAP medium. Imaging was performed with a Zeiss Axio Observer.Z1 microscope (Carl Zeiss S.A.S.) with a X100 oil immersion objective equipped with the filter sets 10 Alexa Fluor (Excitation BP 450/490, Beam Splitter FT 510, Emission BP 515–565), DAPI (Excitation BP 359–371, Beam Splitter FT 395, Emission BP 397-∞), 47 HE CFP (Excitation BP 424/448, Beam Splitter FT 455, Emission BP 460–500), 46 HE YFP (Excitation BP 488–512, Beam Splitter FT 515, Emission BP 520–550) and Chloro (Excitation BP 450/490, Beam Splitter FT 505, Emission BP 600-∞). Cell contours were visualized with Nomarski optics. Images were acquired with an ORCA-R2 charge-coupled device camera (Hamamatsu) and analyzed with ImageJ.
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