SV-589 cells were set up for experiments on day 0 as described in the figure legends. Following incubations described in the figure legends, cells were washed with PBS and subsequently fixed and permeabilized for 15 min in methanol at −20°C. Upon blocking with 1 mg/ml BSA in PBS, coverslips were incubated for 30 min at 37°C with primary antibodies (IgG-H8 against UBIAD1, rabbit polyclonal anti-GM130 IgG [Diao et al., 2003 (link)], and IgG-9E10, a mouse monoclonal antibody against c-Myc purified from the culture medium of hybridoma clone 9E10 [American Type Culture Collection, Manassas, VA]) diluted in PBS containing 1 mg/ml BSA. Bound antibodies were visualized with goat anti-mouse IgG conjugated to Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (Life Technologies, Grand Island, NY) as described in the figure legends. In addition, coverslips were stained for 5 min with 1 µg/ml Hoechst 33,342 (Life Technologies) to visualize nuclei. The coverslips were then mounted in Mowiol 4-88 solution (Calbiochem/EMD Millipore, Billerica, MA) and fluorescence analysis was performed using a Plan-Apochromat 63×/1.4 DIC objective (Zeiss, Peabody, MA), an Axiovert 200M microscope (Zeiss), an Orca 285 camera (Hamamatsu,Houston, TX), and the software Openlab 4.0.2 (Improvision, Coventry, England).
Orca 285 camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The Orca 285 camera is a high-performance, scientific-grade camera developed by Hamamatsu Photonics. It is designed for applications that require high-quality image capture and sensitive detection. The camera features a highly sensitive CCD sensor and advanced readout electronics to provide excellent image quality and data acquisition capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m microscope, by Zeiss (3 mentions)
Openlab 4, by PerkinElmer (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Bisbenzimide hoechst 33342, by Merck Group (2 mentions)
Mz75 light microscope, by Leica (2 mentions)
Axiophot microscope, by Hamamatsu Photonics (2 mentions)
Af488 or cy3 conjugated secondary, by Merck Group (2 mentions)
Bx61 microscope, by Olympus (2 mentions)
Citifluor af2 antifade reagent, by Agar Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Bcl11a, by Abcam (1 mentions)
Anti bcl11a, by Abcam (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
Plan apochromat 63 1.4 objective, by Zeiss (1 mentions)
Lsm 780 inverted microscope, by Zeiss (1 mentions)
7 protocols using orca 285 camera
1
Immunofluorescence Microscopy of UBIAD1
2
Immunostaining of Bcl11a and Related Proteins
3
Immunofluorescence Imaging of Mammary Gland
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Protein samples were prepared as described previously14 (link) and probed using anti-BCL11A (Abcam, Clone 14B5, 1:1000) and Actin (Cell Signalling, 1:10000). For IHC analysis, BCL11A (Abcam (14B5, 1:50); CK14 (Abcam; 1:100) and ERα (SCBT; 1:50) were used. Staining was detected using AF488- or Cy3-conjugated secondary (Sigma) and bisbenzimide-Hoechst 33342 (Sigma). Fluorescence microscopy was carried out using a Zeiss Axiophot microscope equipped with a Hamamatsu Orca 285 camera, with images visualized, captured and manipulated using Simple PCI 6 (C Imaging Systems). The hematoxylin- and eosin-stained samples were visualized on a LEICA light microscope, while the mouse mammary gland whole mounts were visualized using the LEICA MZ75 light microscope.
4
Visualizing Root Surface Carbohydrate Epitopes
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To observe carbohydrate epitopes at root surfaces, 1 to 2 cm regions of whole barley roots with root hairs (WT) or equivalent regions of brb roots were excised and placed overnight in a 4% (w/v) formaldehyde fixative and processed for whole mount immunofluorescence labeling procedures essentially as described (Jackson et al., 2012 ). After antibody incubations, intact root regions were mounted using Citifluor AF2 antifade reagent (glycerol suspension; Agar Scientific Stansted, U.K.) and examined using an Olympus BX-61 microscope with epifluorescence irradiation from a mercury lamp and with excitation (∼480 nm) and emission (∼510 nm) for fluorescein isothiocyanate (FITC) fluorescence detection. Images were captured using a Hamamatsu ORCA285 camera and Volocity software. For each antibody, a manual exposure time was maintained for the capture of all shown micrographs across the two barley genotypes.
5
Immunofluorescence Imaging of Cell Structures
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Cells grown on glass coverslips were fixed and permeabilized in precooled methanol for 15 min at −20°C, washed in PBS, incubated with indicated primary antibodies for 30 min at 37°C, washed, and incubated with Alexa Fluor–conjugated secondary antibodies for 30 min at 37°C. DNA was stained for 5 min with 1 µg/ml Hoechst 33342 (Invitrogen) in PBS at room temperature, and cells were mounted in Mowiol 4–88 (Calbiochem) embedding solution (Wei and Seemann, 2009b (link)). Confocal images were acquired using an LSM780 inverted microscope (Zeiss) with a Plan-Apochromat 63×/1.4 objective (Zeiss) and Zen imaging software (Zeiss). Z-sections were captured at 0.47-µm intervals. All images are confocal maximum-intensity projections assembled using the Zen software package (Zeiss) with the exception of Fig. 5 F . Images shown in Fig. 5 F were captured using an Axiovert 200M microscope (Zeiss) with a Plan-Neofluar 40×/1.3 differential interference contrast objective (Zeiss), an Orca 285 camera (Hamamatsu), and Openlab 4.0.2 software (Improvision).
6
Fluorescence Imaging of Transfected SV589 Cells
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SV589 cells were set up on glass coverslips at 1.5 × 105 cells per 6-well plate in 2 ml medium C with 5% FCS. At 24 hr after plating, cells were transfected with 1 µg of the indicated plasmid using FuGENE HD as the transfection agent. At 24 hr after transfection, cells were fixed for 15 min in 3.7% formaldehyde in PBS at room temperature and permeabilized for 10 min in methanol at −20˚C. After blocking by incubation with 1 mg/ml bovine serum albumin in PBS, cells were double-labeled with 1 µg/ml mouse monoclonal anti-LAMP-2 and 0.8 µg/ml of rabbit monoclonal anti-Flag followed by 6.7 µg/ml goat anti-rabbit IgG conjugated with AlexaFluor 488 and 6.7 µg/ml goat anti-mouse IgG conjugated with AlexaFluor 594. The coverslips were than mounted in Mowiol (EMD, Darmstadt, Germany) solution (Wei and Seemann, 2009 (link)) and fluorescence images were acquired using a Plan-Neofluar 40x/1.3 DIC objective (Zeiss, Oberkochen, Germany), an Axiovert 200M microscope (Zeiss), an Orca 285 camera (Hamamatsu, Hamamatsu City, Japan), and Openlab 4.0.2 software (Improvision, Coventry, UK).
7
Visualizing Carbohydrate Epitopes in Barley Roots
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To observe carbohydrate epitopes at root surfaces, 1 to 2 cm regions of whole barley roots with root hairs (WT) or equivalent regions of brb roots were excised and placed overnight in a 4% (w/v) formaldehyde fixative and processed for whole mount immunofluorescence labelling procedures essentially as described (Jackson et al. 2012) . After antibody incubations intact root regions were mounted using Citifluor AF2 antifade reagent (glycerol suspension; Agar Scientific Stansted, U.K.) and examined using an Olympus BX-61 microscope with epifluorescence irradiation. Images were captured using a Hamamatsu ORCA285 camera and Volocity software. For each antibody, a manual exposure time was maintained for the capture of all shown micrographs across the two barley genotypes.
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