The coding sequence of BcHTT4 was amplified by primers BcHTT4-XbaI-cYFP-F and BcHTT4-KpnI-cYFP-R (Table S1 ) and cloned into pUC-SPYCE vector to generate BcHTT4-cYFP plasmids. The coding sequence of BcFKBP13 was amplified by primers BcFKBP13-XbaI-cYFP-F and BcFKBP13-KpnI-cYFP-R (Table S1 ) and cloned into pUC-SPYNE for generating BcFKBP13-nYFP. The plasmid mixtures (BcHTT4-cYFP and BcFKBP13-nYFP, BcHTT4-cYFP and pUC-SPYNE) were introduced into the tobacco cells. After incubation in the dark for 36 h, the yellow fluorescence signal was observed using an Olympus Fluoview1000 microscope [50 (link)].
Fluoview 1000 microscope
Manufactured by Olympus
Sourced in Japan
The Fluoview 1000 is a confocal microscope system designed for high-resolution imaging. It is capable of capturing detailed, three-dimensional images of samples by scanning the sample with a focused laser beam and detecting the resulting fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ht10132, by Merck Group (2 mentions)
Tunel apogreen detection kit, by Yeasen (2 mentions)
Vectorshield mounting media with dapi, by Vector Laboratories (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Fluoview software, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti cav 1, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Dm6b microscope, by Leica (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by BD (1 mentions)
Facscalibur, by Beckman Coulter (1 mentions)
Quasar 670, by Biosearch Technologies (1 mentions)
Quasar 570, by Biosearch Technologies (1 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
55 protocols using fluoview 1000 microscope
1
Bimolecular Fluorescence Complementation Assay
2
Immunofluorescence Imaging of IP3R1/3 Constructs
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HEK-3KO and HEK-3KO cells stably expressing WT and mutant IP3R1/3 constructs were plated on poly-d -lysine (100 μg/mL)-coated coverslips. At roughly 50% confluent, cells were fixed using 4% parafomaldehyde at room temperature for 10 min. Subsequently, coverslips were washed with PBS, and cells were blocked in 10% bovine serum albumin (BSA) for 1 h. Following blocking, cells were incubated in primary antibody against IP3R3 (BD Transduction) and IP3R1 (#ARC154, Antibody Research Corporation) overnight at 4 °C. The following day, the primary antibody was removed, and coverslips were washed 3 times with PBS for 10 min with gentle rocking. Subsequently, the appropriate secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) was incubated for 1 h at room temperature with gentle rocking. After incubation, coverslips were washed with PBS and mounted on slides. After allowing slides to dry, coverslips were sealed onto slides and imaged using confocal microscopy using an Olympus Fluoview 1000 microscope.
3
Quantification of CAV-1 Expression in Immune Cells
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PBMCs were collected by cytospin and stained using the indicated primary antibodies. Cells were fixed with 2% paraformaldehyde and then permeabilized with 0.1% Triton X-100. Cells were incubated with Alexa Flour® 647-conjugated anti-CD3, CD14, or CD19 (BioLegend, San Diego, CA), and anti-CAV-1 (Santa Cruz Biotechnology), followed by Alexa Flour® 488-conjugated secondary IgG (Invitrogen, Grand Island, NY). Appropriate isotype IgG was used as a control. ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI; Life Technology, Carlsbad, CA) was used for nuclear identification and mounting. The intensity of CAV-1 staining was quantified in randomly chosen CD3+ T cells, CD19+ B cells, and CD14+ monocytes. The data were obtained from 20 cells from each three patients and healthy subjects. Images were taken and analysed using an Olympus Fluoview 1000 microscope (Olympus America, Melville, NY) and fixed camera settings.
4
Whole-mount in situ hybridization of Drosophila brains
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Whole-mount in situ hybridization was performed as described previously (14 (link)). Brains dissected from juvenile and mature wild-type Canton-S males were used for these experiments. smFISH was performed following the published protocol (63 (link)), with the exception that we omitted (i) the overnight incubation in 100% ethanol and (ii) the bleaching steps. Fluorescence-tagged TASK7 (Quasar 570) and Dsx (Quasar 670) probes were designed and manufactured using a commercial source (LGC Biosearch Technologies). Confocal sections were acquired using an Olympus Fluoview 1000 microscope at 3-μm intervals.
5
Transgenic Worm Strain Generation
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Localization constructs were created by cloning a promoter region of 3,464 bp 5′ to the start of the ceh-60 gene and ceh-60 cDNA into a modified pSM vector carrying a GFP reporter sequence preceded by an SL2 trans-splicing site (kindly provided by C. Bargmann, Rockefeller University, New York, NY). The promoter and cDNA sequences were inserted right before the SL2 site using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, Ipswich, MA) and transformed into DH5-alpha competent cells. Purified plasmid DNA was microinjected into the syncytial gonad of young adult worms at 25 ng/μL together with a co-injection marker unc-122p::DsRed at 50 ng/μL. Localization strains were mounted on 2% agarose pads, anesthetized with 1 mM tetramisole, and visualized with a confocal FluoView1000 microscope (Olympus, Japan) or a DM6 B microscope (Leica, Germany).
6
Immunofluorescence Staining Protocol
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Cells were cultured on heat-sterilized coverslips. The cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.1% Triton-X 100 (Sigma) and blocked with 5% bovine serum albumin (Sigma) for 1 h at room temperature. Cells were then incubated with primary antibodies for an overnight at 4 °C. Then, the cells were rinsed with PBS and incubated with secondary antibodies for an hour at room temperature. Cells were imaged using Olympus Fluoview 1000 microscope.
7
Annexin V-FITC Apoptosis Assay
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Apoptosis of transfected cells was detected using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Pharmingen, USA) and analyzed using a FACS Calibur flow cytometry (Beckman FC400 MPL, USA), Annexin V-FITC and propidium iodide (PI) staining. Cells with negative Annexin V and negative PI staining represented normal cells; cells with positive Annexin V and negative PI represented cell apoptosis; cells with positive Annexin V and positive PI staining represented cell necrosis. Fluorescence results of stained cells were also obtained via the Olympus FluoView™ 1000 microscope (CME-UFRGS). The assays were performed in triplicate.
8
Dissection and smFISH Analysis of Drosophila Brains
9
Mitochondrial Imaging and Protein Localization
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Cells were cultured on the coverslips and stained with MitoTracker DeepRed FM (Cellsignaling Technology) to indicate mitochondria according to the manufacture’s protocol. Then, cells were permeabilized with 0.25%Triton X-100/PBS. Post 30 min, cells were incubated with 0.5% BSA for 1 h and primary antibodies overnight. Then, cells were washed and incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen). The nuclear were counterstained using DAPI. High-resolution images were captured using the Olympus Fluoview 1000 microscope.
10
Histological Analysis of Testes and Epididymis
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Testes and epididymis from at least three mice for each genotype were collected when the mice were killed after cervical dislocation and fixed in Bouin’s solution (Sigma, HT10132) at 4 °C overnight. Then, the samples were dehydrated stepwise by using an ethanol series (70%, 90%, and 100% ethanol), embedded in paraffin, and cut into 5 µm tissue slices. After dewaxing and hydration, the sections were stained with periodic acid-Schiff (PAS) and imaged with a FluoView 1000 microscope (Olympus, Tokyo, Japan) with a digital camera (MSX2, Micro-shot Technology Limited, Guangzhou, China).
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