Confocal images were taken with a LSM510 scanning confocal microscope (Zeiss, Thornwood, NY). Laser excitation was at 488 nm and for fluorescence emission detection, a 520/40 BP filter (all organelle stains) and a 620/20 BP filter (QDots or PDots) were utilized. Colocalization of QDots and PDots with organelles was undertaken using Metamorph image analysis program (Molecular Devices, Sunnyvale, CA).
Metamorph image analysis program
Manufactured by Molecular Devices
MetaMorph is an image analysis software program used to acquire, analyze, and manage digital images obtained from microscopy. It provides tools for image processing, measurement, and quantification of cellular and subcellular features.
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Lab products found in correlation
Lsm510 scanning confocal microscope, by Zeiss (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Bx61 32fdic, by Olympus (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
2 protocols using metamorph image analysis program
1
Confocal Imaging of Nanoparticle Colocalization
2
Papiliocin Inhibits LPS-Induced NF-κB Activation
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The 5 × 105 RAW 264.7 cells were seeded on the coverslips in 6-well plates. After 16 h, cells were pretreated with 10 μM papiliocin for 1 h before 50 ng/mL LPS treatment. After 30 min, the cells were fixed by 4% paraformaldehyde and permeabilized by 0.3% Triton X-100. After blocking (5% BSA), primary antibodies against p-NF-κB (Cell Signaling Technology, No. 3031, 1:50) or α-tubulin (Cell Signaling Technology, No. 3873, 1:200) were added for 90 min and then incubated with Alexa Fluor 488 (Invitrogen, No. A-28175) or Alexa Fluor 546 (Invitrogen, No. A-10040, 1:200) conjugated secondary antibodies for 40 min. We counterstained the nuclear region using Hoechst 33258 (Invitrogen, No. H3569, 1:200) and visualized by fluorescence microscope (BX61-32FDIC, Olympus) and analyzed by the MetaMorph Image analysis program (Molecular Devices).
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