Immunofluorescence was performed as described [47 (link)] using antibodies against Podocalyxin (catalog # PA1-46169; Thermo Fisher Scientific) and Syntaxin-4 (catalog # 610439; BD Biosciences; San Jose, CA). Confocal analyses were performed using an Olympus FV3000 confocal microscopy system (Olympus; Waltham, MA).
Fv3000 confocal microscopy system
Manufactured by Olympus
The FV3000 confocal microscopy system is a high-performance imaging solution designed for advanced fluorescence microscopy. It features a high-resolution optical system, sensitive detectors, and a modular design to enable flexible configurations for a wide range of applications. The FV3000 provides researchers with powerful tools for visualizing and analyzing complex biological samples at the cellular and subcellular levels.
Automatically generated - may contain errors
Lab products found in correlation
Podocalyxin, by Thermo Fisher Scientific (1 mentions)
Fluorescence stereomicroscope, by Leica (1 mentions)
Smz18 fluorescence stereomicroscope, by Nikon (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Airyscan, by Zeiss (1 mentions)
Rabbit anti cd11b, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
5 protocols using fv3000 confocal microscopy system
1
Podocalyxin and Syntaxin-4 Visualization
2
Multimodal Microscopic Imaging Protocol
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Immunostained images, blood cell images, and wing Disc images were acquired using Olympus FV3000 confocal microscopy system under a × 20 air or × 40 oil-immersion objective or × 60 oil-immersion objective. Bright-field and larval fluorescence images were obtained on Leica fluorescence stereomicroscope.
3
Screening Lysosomal Regulators in C. elegans
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The primary screen was performed on 95 lysosomal-enriched candidates shared between the LMP-1 and the CTNS-1 Lyso-IP proteomic profiling datasets. Each RNAi bacteria clone was seeded onto 12-well RNAi plates containing 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and allowed to dry. The dried plates were then incubated at room temperature overnight to induce dsRNA expression. Synchronized L1 nre-1(hd20);lin-15b(hd126) worms were seeded on 12-well RNAi plates and raised at 20°C for 2 days, and then around 30 worms each well were transferred to the RNAi plates containing RNAi bacteria and 0.5 μM of LysoSensor Green DND-189. After 18 hr, LysoSensor signals were examined by the naked eyes using a Nikon SMZ18 fluorescence stereo microscope. The candidates with obvious LysoSensor alteration were selected for the secondary LysosSensor RNAi screen. In the secondary screen, worms stained using LysoSensor Green DND-189 were imaged by the Olympus FV3000 confocal microscopy system. The changes in LysoSensor signals in the first pair of intestine cells were quantified by ImageJ (including intensity, size, and number).
4
Tissue-specific CRISPR Localization Imaging
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Tissue-specific lyso-tag expression example images (Figure 3A ) were captured using Leica DMi8 THUNDER Imaging Systems using ×20 objective. The images that show the colocalization between CRISPR knock-in lines LMP-1::mNeonGreen and CTNS-1::wrmScarlet were captured by Zeiss LSM 980 with Airyscan. The images that show the colocalization between wrmScarlet::LMTR-3 and LMP-1::mNeonGreen/CTNS-1::mNeonGreen were taken using Nikon CSU-W1 spinning disk confocal microscopy system. Other microscopy images were captured using an Olympus FV3000 confocal microscopy system using ×60 or ×20 objective. C. elegans were anesthetized in 1% sodium azide in M9 buffer and placed on a 2% agarose pad sandwiched between the glass microscopic slide and coverslip.
5
Immunofluorescence Staining of Frozen Tissue Sections
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Immunofluorescence staining was performed on frozen sections cut into 4-μm slices, blocked and permeated with 3% bovine serum albumin–0.5% Triton X-100 (Sigma) for 30 minutes at room temperature, followed by incubation with rabbit anti-CD11b (1:100, 17800; Cell Signaling Technology, Danvers, MA) and rat anti–Ly-6G (1:200, #88876; Cell Signaling Technology) overnight at 4°C. Nuclei were stained with 4’,6-diamidino-2-phenylindole dihydrochloride. Then, slides were washed with phosphate-buffered saline and incubated with horseradish-peroxidase–conjugated secondary antibodies. Confocal microscopy was performed using an Olympus FV3000 confocal microscopy system.
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