P-gp transport activity levels and P-gp-mediated Aβ transport were determined as previously described [9 (link), 28 (link), 29 (link)]. To determine P-gp transport activity, freshly isolated brain capillaries were incubated with 2 µM of the P-gp-specific substrate NBD-cyclosporin A (NBD-CSA) in DPBS for 1 hour. To assess P-gp-mediated hAβ42 transport activity, brain capillaries were incubated with 5 µM fluorescein-hAβ42 in DPBS for 1 hour. Images of 10 capillaries per treatment group were captured by confocal microscopy using the 488 nm line of an argon laser of a Leica TCS SP5 confocal microscope with a 63 × 1.2 NA water immersion objective (Leica Instruments, Wetzlar, Germany). NBD-CSA fluorescence in the capillary lumen was measured in each image using Image J v.1.48v (Wayne Rasband, NIH, USA; RRID:SCR_003070). Specific, luminal NBD-CSA or fluorescein-hAβ42 fluorescence were measured as the difference between total luminal fluorescence and fluorescence in the presence of 5 µM PSC833, a P-gp-specific inhibitor [9 (link), 28 (link), 29 (link)].
1.2 na water immersion objective
Manufactured by Leica
Sourced in Germany
The 63 × 1.2 NA water immersion objective is a high-numerical aperture (NA) objective designed for use with Leica microscopes. It features a magnification of 63× and a numerical aperture of 1.2, which allows for high-resolution imaging with a large field of view. This objective is optimized for use with water as the immersion medium.
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Lab products found in correlation
Tcs sp5 confocal microscope, by Leica (2 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Rm2125 rotary microtome, by Leica (1 mentions)
Dmi6000b, by Leica (1 mentions)
Orca ag camera, by Hamamatsu Photonics (1 mentions)
Bx50wi upright microscope, by Hamamatsu Photonics (1 mentions)
5 protocols using 1.2 na water immersion objective
1
Measuring P-gp Activity and Aβ Transport
2
Immunofluorescence Staining Protocol
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For immunofluorescence, cells were grown on cover slips, washed with 1× PBS, and fixed in 3% paraformaldehyde for 10 min at room temperature. Afterward, cells were washed with 1× PBS and blocked using 1× PBS containing 0.1% Triton X-100 and 10% FCS (PBSTS). Fixed cells were incubated with primary antibodies for 1 h, washed, and then incubated with secondary antibodies for 1 h. Primary and secondary antibodies were diluted in 1× PBSTS (primary: α-SMN 7B10, mouse, from 1.5 µg/ml 1:700, α-coilin H300 rabbit [sc-32860; Santa Cruz Biotechnology, Heidelberg, Germany] from 1 μg/ml 1:200, α-coilin F-7 mouse [sc-55594; Santa Cruz Biotechnology] from 1 μg/ml 1:500, α-PTPN23, rabbit, from 1 μg/ml 1:100). DNA was stained using DAPI at a concentration of 0.25 μg/ml. Cells were washed and mounted on object slides using Fluoromount-G mounting medium (Southernbiotech, Birmingham, AL). Analysis was done on Leica SP2 confocal microscope using a 63×/1.2 NA water immersion objective (Leica Microsystems, Mannheim, Germany). Images were processed using the Leica confocal software and Photoshop CS4.
3
Aβ Transport Regulation in Capillaries
4
Visualizing Arabidopsis Leaf Immunity
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Leaves of A. thaliana wild-type (Col-0) or the shrk1 x shrk2 double mutant were harvested at 5 dpi, cleared in 10% KOH for 5 min, stained with 0.05% aniline blue in 67 mM K2HPO4 for 20 min and observed with a Leica SP5 confocal light scanning microscope. Images were edited using ImageJ with the “volume viewer” plugin [77 (link)].
For Technovit sections, A. thaliana wild-type (Col-0) or mutant leaves were infected with Hpa as described above and harvested at 7 dpi. Leaves were fixed with 3.7% formaldehyde and dehydrated by incubating samples in 30%, 50%, 70% and 100% ethanol. Samples were embedded in Technovit 7100 according to the manufacturer’s instruction. A Leica RM2125 rotary microtome was used to cut 7μ m sections. Sections were stained in 0.01% trypan-blue-lactophenol for 3 h at 37°C, followed by clearing in chloral hydrate (2.5g/ml) and subsequent differential interference contrast microscopy with a Leica DMI6000B.
A. thaliana wild-type (Col-0) plants expressing RPW8.2-YFP [38 (link)] were infected with Hpa as described above, harvested at 10 dpi and observed with a Leica TCS SP 5 confocal laser scanning microscope equipped with a 63x NA 1.2 water-immersion objective (excitation with an Argon laser 514 nm, detection at 520–560 nm).
For Technovit sections, A. thaliana wild-type (Col-0) or mutant leaves were infected with Hpa as described above and harvested at 7 dpi. Leaves were fixed with 3.7% formaldehyde and dehydrated by incubating samples in 30%, 50%, 70% and 100% ethanol. Samples were embedded in Technovit 7100 according to the manufacturer’s instruction. A Leica RM2125 rotary microtome was used to cut 7
A. thaliana wild-type (Col-0) plants expressing RPW8.2-YFP [38 (link)] were infected with Hpa as described above, harvested at 10 dpi and observed with a Leica TCS SP 5 confocal laser scanning microscope equipped with a 63x NA 1.2 water-immersion objective (excitation with an Argon laser 514 nm, detection at 520–560 nm).
5
Visualizing Ca2+ Dynamics in Live Trophoblasts
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Kinetic of annexin fusion-tagged protein translocation to plasmalemma was monitor in live cells or fixed trophoblasts using a laser scanning confocal microscope (SP5; Leica) equipped with a 63x N.A. 1.2 water-immersion objective in the line-scan mode. Trophoblasts were incubated with ionomycin (10 μM). For live cells, images were acquired before incubation and automatically within 20 s intervals for 10 min after incubation.
Optical recording of intracellular Ca2+ transient was performed by loaded trophoblasts with the membrane-permeant Fluo-4 AM as recommended by the manufacturer protocol. Wide-field images were obtained with Olympus BX50WI upright microscope with a 40 × 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu)50 (link). Images were acquired automatically within 60 s intervals. Pseudo-color images display the intracellular Ca2+ value coded in hue and the fluorescence intensity coded in intensity.
Optical recording of intracellular Ca2+ transient was performed by loaded trophoblasts with the membrane-permeant Fluo-4 AM as recommended by the manufacturer protocol. Wide-field images were obtained with Olympus BX50WI upright microscope with a 40 × 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu)50 (link). Images were acquired automatically within 60 s intervals. Pseudo-color images display the intracellular Ca2+ value coded in hue and the fluorescence intensity coded in intensity.
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