Western blot analysis was performed as described previously (Li et al., 2012 (link)). Image acquisition and quantitation of band intensity were performed using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). For immunoprecipitation, the cells were lysed in buffer (50mM Tris·HCl, pH 8.0, 150mM NaCl, 5mM EDTA, and 0.5% Nonidet P-40) and centrifuged at 16,000 × g for 30 minutes to remove debris. Cleared lysates were subjected to immunoprecipitation with antibodies. For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes, permeabilized in 5% Triton X-100 for 5 minutes, and then stained using primary antibodies. The secondary antibodies used were mouse Alexa Fluor 488 or 594 dye conjugate, or rabbit Alexa Fluor 488 or 594 dye conjugate (Thermo Fisher Scientific). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI blue; Thermo Fisher Scientific). After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss).
4 6 diamidino 2 phenylindole dapi blue
Manufactured by Thermo Fisher Scientific
4′,6-diamidino-2-phenylindole (DAPI) is a fluorescent stain that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used in fluorescence microscopy and flow cytometry applications.
Automatically generated - may contain errors
2 protocols using 4 6 diamidino 2 phenylindole dapi blue
1
Immunoprecipitation and Immunocytochemistry Protocols
2
Visualizing CD3/CD28 Distribution in Jurkat T Cells
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Monolayers of Jurkat T cells were fixed on a glass-bottom petri dish (TF) with 4% formaldehyde (TF) for 20 min at RT. The cells were washed 3X with 5% Tween 20 (TF) in PBS and blocked with 1% BSA in PBS for 1 h at RT. After blocking, the cells were again washed 3X with 5% Tween 20 in PBS and incubated with anti-CD3 (clone PC3/188A)48 (link) and anti-CD28 (clone CD28.2)49 (link) conjugated to Alexa Fluor 488 (green; 1:200 dilution; SCB) and Allophycocyanin (APC; red; 1:200 dilution; SCB), respectively, in blocking solution in an orbital shaker (Eppendorf Galaxy 170S) at 150 rpm and 37 °C for 1 h. The samples were washed 3X with blocking buffer and incubated with 4′, 6-diamidino-2-phenylindole (dapi; blue; Thermo Fisher; TF) to stain nuclei for 1 h at 37 °C. The slides were washed 3X with PBS, coverslips were affixed, and each sample was viewed to determine the CD3/CD28 distribution on the surface of Jurkat T cells with a Zeiss LSM 700 Confocal Laser Scanning Microscope (CZ) at a magnification of 40 × . Images were collected using an AxioCam digital camera and analyzed using Zen Lite digital imaging software (CZ). This fluorescent microscopy methodology is a modification of a similar and previously described procedure50 (link).
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