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).
Lsm510 scanning confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM510 is a scanning confocal microscope manufactured by Zeiss. It is designed to provide high-resolution optical sectioning and three-dimensional imaging of biological samples. The instrument utilizes laser illumination and a detection system to selectively capture light from a specific focal plane within the specimen, allowing for the elimination of out-of-focus information and improved image clarity.
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Lab products found in correlation
Metamorph image analysis program, by Molecular Devices (1 mentions)
μ slide vi0, by Ibidi (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
3 protocols using lsm510 scanning confocal microscope
1
Confocal Imaging of Nanoparticle Colocalization
2
Biofilm Formation of Pseudomonas sp. UK4
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One colony of Pseudomonas sp. UK4 (wt, ∆fap, and pfap) was transferred to the LB medium for overnight growth at 28 °C, 180 rpm, diluted to OD600~0.5, and transferred to an IBIDI® μ-slide VI0.4 uncoated channel slide. After 1 h incubation, the bacteria were gently washed with fresh LB medium, after which the LB medium with 100 µM of PGG and EGCG was circulated through the chambers at a flow rate of 2 mL/h for 48 h (Harvard PHD 2000 Infuse/Withdraw Syringe Pump) to enable formation of biofilm under dynamic conditions. After washing the chambers with PBS and staining with live/dead kit using Cyto9 and propidium iodide, bacteria were imaged with LSM 510 scanning confocal microscope (Zeiss GmbH, Jena, Germany).
3
RN2N Delivery and Brain Uptake
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RN2N was conjugated with Alexa Fluor® 647 (Thermo Fisher) and purified by size exclusion chromatography using a Superdex 200 10/300 column (GE Healthcare) equilibrated in PBS pH 7.4 at 0.5 ml/min.
pR5 mice aged 6 or 8 months were randomly assigned to the following groups: SUS only, RN2N only and combined SUS and RN2N. Three mice were used per group and treatment was conducted as described above. Different from above, to assess antibody delivery pR5 mice were injected with a higher dose of 90 μg RN2N. At 30 min after delivery, mice were given an overdose of sodium pentabarbitone and transcardially perfused with PBS. The brains were dissected as described above. For analysis of brain uptake of Alexa Fluor® 647-conjugated RN2N, the fixed hemispheres of the mice were imaged in a Li-COR Odyssey Fc near-infrared scanner (700 nm laser 2 min scan time) before being cryo-protected by immersion in 30% sucrose and then sectioned at 40-μm thickness on a freezing sliding microtome. For immunofluorescence, sections were incubated with primary antibodies against tau (HT7), EEA1 and LAMP1 overnight, followed by 2 h incubation in Alexa Fluor® 488-conjugated secondary antibodies. Sections were mounted on slides and stained with 1 μg/ml DAPI in PBS for 10 min, and cover-slipped with fluorescent mounting medium (Dako). Images were taken with a Zeiss LSM510 scanning confocal microscope.
pR5 mice aged 6 or 8 months were randomly assigned to the following groups: SUS only, RN2N only and combined SUS and RN2N. Three mice were used per group and treatment was conducted as described above. Different from above, to assess antibody delivery pR5 mice were injected with a higher dose of 90 μg RN2N. At 30 min after delivery, mice were given an overdose of sodium pentabarbitone and transcardially perfused with PBS. The brains were dissected as described above. For analysis of brain uptake of Alexa Fluor® 647-conjugated RN2N, the fixed hemispheres of the mice were imaged in a Li-COR Odyssey Fc near-infrared scanner (700 nm laser 2 min scan time) before being cryo-protected by immersion in 30% sucrose and then sectioned at 40-μm thickness on a freezing sliding microtome. For immunofluorescence, sections were incubated with primary antibodies against tau (HT7), EEA1 and LAMP1 overnight, followed by 2 h incubation in Alexa Fluor® 488-conjugated secondary antibodies. Sections were mounted on slides and stained with 1 μg/ml DAPI in PBS for 10 min, and cover-slipped with fluorescent mounting medium (Dako). Images were taken with a Zeiss LSM510 scanning confocal microscope.
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