Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in blocking buffer at 4°C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen H3570). Fluorescence was visualized with Leica DMi8 microscope with ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: Olig2 (Millipore MABN50), RFP (Rockland 600–401-379), donkey anti-mouse IgG, Alexa Fluor 488 (ThermoFisher A21202) and goat anti-rabbit IgG, Alexa Fluor 594 (ThermoFisher A11037).
Orca flash4.0 scmos camera
Manufactured by Leica
The ORCA-Flash4.0 sCMOS camera is a high-performance scientific CMOS (sCMOS) camera designed for a wide range of applications in life science research and industrial imaging. It features a large sensor size, high resolution, and fast frame rates, making it suitable for a variety of imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
Dmi8 microscope, by Leica (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse calnexin antibody, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Dmi6000 inverted microscope, by Hamamatsu Photonics (1 mentions)
Mab386, by Merck Group (1 mentions)
Ab290, by Merck Group (1 mentions)
Ab9106, by Abcam (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
H3570, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A11058, by Thermo Fisher Scientific (1 mentions)
7 protocols using orca flash4.0 scmos camera
1
Immunofluorescence Staining of Olig2 and RFP
2
Immunofluorescent Protein Localization
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Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in the blocking buffer at 4 °C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: monoclonal anti-Flag M2 antibody (Sigma, 1:1000), goat anti-mouse calnexin antibody (Santa Cruz, 1:500), donkey anti-mouse antibody, Alexa Fluor 488 conjugate (Thermo Fisher, 1:5000), and donkey anti-goat antibody, Alexa Fluor 594 conjugate (ThermoFisher, 1:5000).
3
Quantitative Analysis of CRISPR-Mediated Oligodendrocyte Differentiation
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DNA plasmids that express dCas9-KRAB and sgRNAs were transfected into primary mouse OPCs using Lipofectamine 2000. Transfected OPCs were kept in the differentiation condition for 3 days. Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in the blocking buffer at 4 °C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Images were taken in a blind manner. The signal from each fluorescence channel (Hoechst, GFP, and RFP) was quantified by CellProfiler for an objective quantitative analysis.
4
Matrigel-Based 3D Acini Assay
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Lab-Tek II plates (Lab-Tek II, #155409) were coated with 20 μl Matrigel (BD Biosciences, 356231). A total of 1,000 cells were suspended in 100 μl Matrigel and overlaid in wells. After polymerization at 37 °C, 500 μl of cell culture media was added and replaced every 3 days. Cells were grown for 14 days and treated with cell culture medium containing serum, growth factors and EtOH or 4OH-TAM. Acini were imaged by transmitted light and DIC optics for maximum contrast using a Leica DMI6000 inverted microscope coupled to a Hamamatsu Orca Flash 4.0 sCMOS camera with a Leica 10 × 0.3 numerical aperture PLAN FLUOR dry objective. Image processing and analysis were performed using Fiji (ImageJ package distribution). For size quantification, acini edges were outlined using the Find Edges function, converted to a binary mask before measurement using the area function.
5
Immunofluorescence Staining of Cellular Proteins
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Cells were cultured on coverglass and fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in blocking buffer at 4°C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: monoclonal ANTI-FLAG® M2 antibody (Sigma #F1804, 1:1000), anti-Myc tag (Abcam #ab9106, 1:1000), anti-GFP (Abcam #ab290, 1:5000), anti-MBP (Millipore #MAB386, 1:500), goat anti-rabbit, Alexa Fluor® 594 conjugate (ThernoFisher #A11037, 1:10 000), donkey anti-mouse, Alexa Fluor® 488 conjugate (ThernoFisher # A21202, 1:10 000), goat anti-rat, Alexa Fluor® 594 conjugate (ThernoFisher #A21209, 1:10 000), and donkey anti-rabbit, Alexa Fluor® 488 conjugate (ThernoFisher # A21206, 1:10 000).
6
Immunofluorescence Labeling of Cellular Proteins
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Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in blocking buffer at 4 °C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: FLAG antibody (Sigma #F1804, 1:1000), calnexin antibody (Santa Cruz #SC-6465, 1:500), Hoechst 33342 (ThermoFisher #H3570, 1:20000), donkey anti-mouse antibody, Alexa Fluor® 488 conjugate (ThermoFisher #A21202, 1:5000), and donkey anti-goat antibody, Alexa Fluor® 594 conjugate (ThermoFisher #A11058, 1:5000).
7
Immunofluorescence Staining of Cells
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Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in the blocking buffer at 4 °C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: monoclonal ANTI-FLAG M2 antibody (Sigma, 1:1000), goat anti-mouse calnexin antibody (Santa Cruz, 1:500), donkey anti-mouse antibody, Alexa Fluor 488 conjugate (ThermoFisher, 1:5000), and donkey anti-goat antibody, Alexa Fluor 594 conjugate (ThermoFisher, 1:5000).
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