HAoECs were seeded onto sterile eight-well chamber slides (Sigma, C7182) at a concentration of 3 × 104 cells/well. Once the cells were confluent (~80%) in 3—4 days, the culture medium was removed, and the monolayers were washed with warm Dulbecco’s phosphate-buffered saline (DPBS). The cells were then fixed with 4% paraformaldehyde (Sigma, 158127) for 20 min at room temperature followed by permeabilization using 0.1% saponin (Sigma, 47036) for 5 min. The cells were then blocked using 1% bovine serum albumin in DPBS for 1 h at room temperature. After blocking, the cells were washed again with DPBS and incubated with the suitable primary antibody dilution overnight at 4 °C in a humidified chamber. The following day, cells were washed with DPBS, and hereafter, the chamber slides were kept in the dark. The cells were then incubated with suitable fluorescence-tagged secondary antibodies (1:500 dilution) at room temperature for 1—2 h. Following incubation, cells were washed with DPBS and stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma, #10236276001) at 1:10,000 dilution for 5 min. Cells were washed with DPBS, and the chamber was removed from the slide. The slide was then mounted with a clean glass coverslip using a fluoromount mounting medium (Sigma, F4680). Immunofluorescence was detected using a Nikon A1R-Ti2 confocal system.
A1r ti2 confocal system
Manufactured by Nikon
Sourced in United States
The Nikon A1R-Ti2 confocal system is a high-performance microscope designed for advanced imaging applications. It features a resonant scanner for rapid image acquisition and a tilted-illumination system for improved optical sectioning. The system is equipped with a sensitive gallium arsenide phosphide (GaAsP) detector and supports a variety of laser lines, allowing for versatile fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Exoglow protein ev labeling kit, by System Biosciences (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
F4680, by Merck Group (1 mentions)
C7182, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ag25b0006c100, by Thermo Fisher Scientific (1 mentions)
Exoglow rna ev labeling kit, by System Biosciences (1 mentions)
Exoglow vivo ev labeling kit, by System Biosciences (1 mentions)
Evos fl microscope, by Thermo Fisher Scientific (1 mentions)
7 protocols using a1r ti2 confocal system
1
Immunofluorescence Staining of Endothelial Cells
2
ASC Oligomerization and Speck Assay
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ASC speck staining and ASC oligomerization assay were carried out as described (Chen et al., 2019 ). The primary anti‐ASC antibody (Adipogen, San Diego, CA, USA, AG25B0006C100, 1:200) and Alexa‐Fluor‐594‐conjugated secondary antibody (Invitrogen, A‐11037, 1:200) were used. Cell images were obtained using an A1R‐Ti2 confocal system (Nikon).
3
Immunocytochemistry for ZIKV Detection
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After 48 h of ZIKV infection, the media was aspirated and the cells were washed with phosphate buffered saline (PBS). Fixing was done with methanol and acetone at the ratio of 1:1 and washed thrice with PBS. Primary antibody, anti-flavivirus group antigen antibody (D1-4G2-4-15 clone) was used at a dilution of 1:500 and incubated at room temperature for 2 h with gentle rocking. After primary antibody incubation, cells were washed thrice with PBS in 5 min intervals. Alexaflour-488 conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA, USA) was added at a dilution of 1:1000 and kept in a shaker at room temperature for 1 h. After incubation, the cells were washed thrice in PBS and then visualized under Nikon A1R-Ti2 confocal system.
4
Labeling and Tracking GC-VLNs in Cells and Mice
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The lipophilic dye DiI (2 μM) was gently mixed with GC-VLNs in PBS for 30 min at 37 ºC to label the membrane lipids. 35-fold more PBS was added to the mixture, and it was subjected to ultracentrifugation at 100,000 ×g for 2 h at 4 ºC to remove free dye. The obtained GC-VLNs were resuspended in PBS and incubated at 37 ºC for 1 h, ready to be incubated with BMDMs. Proteins and RNAs inside GC-VLNs were labeled with ExoGlow protein EV labeling kit and ExoGlow RNA EV labeling kit (System Biosciences), respectively, per manufacturer's protocols. BMDMs were incubated with the fluorescence-labeled GC-VLNs for 16 h or the indicated time in the time-course experiment, washed with PBS 4 times, and fixed with 4% paraformaldehyde (Sigma). Images were taken using an A1R-Ti2 confocal system (Nikon, Melville, NY, USA).
For the distribution studies of GC-VLNs in mice, fluorescence dye from ExoGlow-Vivo EV labeling kit (EXOGV900A-1, System Biosciences) was used to label GC-VLNs at the ratio of 60×1010 nanoparticles : 1 μl dye, per manufacturer's protocol. The labeled GC-VLNs were resuspended in 30 mL of PBS and ultra-centrifuged at 100,000 ×g for 2 h at 4 ºC to remove the free dye. The wash step was repeated two times. The obtained GC-VLNs covalently linked to the dye were given to mice through oral gavage or intravenous injection.
For the distribution studies of GC-VLNs in mice, fluorescence dye from ExoGlow-Vivo EV labeling kit (EXOGV900A-1, System Biosciences) was used to label GC-VLNs at the ratio of 60×1010 nanoparticles : 1 μl dye, per manufacturer's protocol. The labeled GC-VLNs were resuspended in 30 mL of PBS and ultra-centrifuged at 100,000 ×g for 2 h at 4 ºC to remove the free dye. The wash step was repeated two times. The obtained GC-VLNs covalently linked to the dye were given to mice through oral gavage or intravenous injection.
5
Apoptosis Detection in Liver Sections
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The paraffin-embedded liver sections on the slides were dewaxed by heating at 60 °C for 2 h, followed by 3 rounds of washing in the clearing agent Xylene Substitute (Electron Microscopy Sciences, Hatfield, PA, USA, 23412-01). The samples were rehydrated by sequentially immersing the slides for 5 min in descending concentrations of ethanol (100%, 95%, 50%, 70%, and 30%). The slides were placed in preheated 0.1 M citrate buffer (pH 6) for 15 min at boiling temperature to retrieve the antigen, immersed in running tap water for 10 min, and incubated in PBS for 10 min. The sections were stained using In Situ Cell Death Detection Kit, TMR red (Roche, Indianapolis, IN, USA, 12156792910), which detects single- and double-stranded DNA breaks occurring at the early stages of apoptosis. The fluorescence signals at the damaged sites of the DNAs were obtained using an A1R-Ti2 confocal system (Nikon, Melville, NY, USA).
6
Cellular Localization of IRF3 and I73R
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Hela cells cultured in 4-well culture chambers (BD Biosciences, San Jose, CA, USA) were transfected with 500 ng of pCI-I73R-Flag and 200 ng pCI-IRF3-GFP for 24 h. The cells were then stimulated by transfection with 500 ng poly(dA:dT). Twelve hours later, the cells were fixed in 4 % paraformaldehyde for 10 min at room temperature, followed by permeabilization in 0.5 % Triton X-100 at room temperature for 15 min. The cells were incubated with the anti-Flag antibodies at 4 °C overnight, followed by the Goat Anti-Mouse IgG H+L (Alexa Fluor® 488) for 1 h at room temperature. Subsequently, the cells were counterstained with DAPI (Thermo Scientific, Waltham, MA, USA) for 5 min. The cells were visualized with a Nikon A1R-Ti2 confocal system (Nikon, Melville, NY, USA).
7
Zika Virus Infection in Placental Trophoblasts
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Placental trophoblast was infected with ZIKV, 0.1 MOI for 48–72 h. Infected cell media was aspirated and fixed with methanol and acetone (ratio of 1:1) and washed with PBS. Blocking was done using solution containing 1% BSA and 22.52 mg/ml glycine in PBST. Zika virus envelope protein (E protein) rabbit polyclonal was used at a dilution of 1:500 or CHOP mouse monoclonal antibody at a dilution of 1:400 was used and incubated overnight at 4 °C. After primary antibody incubation, cells were washed thrice with PBS with an interval of 5 mins each. Alexa flour 488/594 conjugated secondary antibody was added at a dilution of 1:1000 and kept in a shaker at room temperature for 2 h. After incubation, the cells were washed thrice in PBS, counter stained with DAPI (0.3 µM) for 10 min at 37 °C and washed with PBS. Images were taken using EVOS FL microscope (Thermo Fisher) or Nikon A1R-Ti2 confocal system.
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