Cells infected with ASFV at a multiplicity of infection (MOI) of 1 were seeded on a 24-well plate and incubated with ASFV p30 protein monoclonal antibody, which was previously diluted with 2% bovine serum albumin (BSA) at a ratio of 1:500. At 24 h post-infection, the cells were washed five times with PBS (1 mL each time), fixed in 500 μL 3.7% paraformaldehyde for 30 min at 25°C, permeabilized in 1 mL 0.1% (w/v) Triton X-100 for 20 min at 25°C, and then incubated in the dark with a secondary antibody diluted with 2% BSA (1:200) for 1 h at 37°C in a humid chamber. Thereafter, cell nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI) at 25°C for 5 min and washed thrice with PBS. Cell fluorescence was observed using an immunofluorescence microscope (Nikon, Tokyo, Japan).
Immunofluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Immunofluorescence microscope is a specialized laboratory instrument used for the visualization and analysis of cellular and molecular structures labeled with fluorescent dyes. It utilizes the principles of immunofluorescence, where specific antibodies conjugated with fluorescent markers are used to target and identify proteins or other biomolecules within a sample. The microscope is equipped with the necessary optics, illumination, and detection systems to capture and analyze the fluorescent signals, providing researchers with a powerful tool for studying cellular processes, protein localization, and molecular interactions.
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Lab products found in correlation
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Nikon (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Nikon (1 mentions)
Cold methanol, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Anti prox1, by R&D Systems (1 mentions)
Anti von willebrand factor vwf, by Agilent Technologies (1 mentions)
Anti ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Confocal laser microscope system, by Nikon (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Pkh67 green fluorescent cell linker mini kit, by Merck Group (1 mentions)
Eclipse te200 microscope, by Nikon (1 mentions)
Exosome free serum, by Thermo Fisher Scientific (1 mentions)
Pkh26, by Merck Group (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
15 protocols using immunofluorescence microscope
1
Quantification of ASFV p30 Protein
2
Immunofluorescence Analysis of HFL-1 Differentiation
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HFL-1 cells were cultured in 6-well-plate for 24–72 hrs, and immunofluorescence was performed to assess HFL-1 differentiation. Cells were fixed with 2% paraformaldehyde for 10 minutes at room temperature, and then washed 3 times 5 minutes each with PBS, permeabilized with 0.5% Triton X-100 PBS for 15 minutes, further washed 3 times, 5 minutes each with PBS, and blocked with 0.3 M glycine, 0.1% Triton X-100, and 5% goat serum in PBS for 1 hr. The cells were incubated with α-SMA (Alexa Fluo-568) primary antibody dilution (1:1000) and fluorescein phalloidin dilution (1:500) in 1% BSA, 0.1% Triton X-100 in PBS overnight at 4 °C, washed 3 times 5 minutes each with PBS, and incubated with donkey anti-mouse secondary antibody dilution (1:1000) in 1% BSA, PBS for 1 hr. Cells were further washed 3 times 5 minutes each with PBS; and mounted with coverslips using 10 μl mounting reagent containing DAPI. Slides were dried for 5 minutes, and the images were taken by immunofluorescence microscope (Nikon).
3
Measuring Cytoplasmic and Mitochondrial ROS
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CM-H2DCFDA (Thermo Scientific) and MitoSOX Red (Thermo Scientific) staining were used for detecting cytoplasmic and mitochondrial ROS levels, respectively. Cells were seeded in 24-well plates and cultured in normoxia or hypoxia for 24 h. Subsequently, cells were incubated with 10 μM CM-H2DCFDA for 30 min or 5 μM MitoSOX Red for 10 min at 37°C, and fluorescence was captured by immunofluorescence microscope (Nikon, Tokyo, Japan) at 20 × magnification. Immunofluorescence density was quantified by Image J software (U. S. National Institutes of Health, MD, United States) and normalized by the siControl normoxia group.
4
Identification of Vascular Cell Types
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The VM-ECs were identified by immunofluorescence and western blot. Human umbilical vein endothelial cells (HUVECs) and human uterine smooth muscle cells (HUSMCs) were identified by immunofluorescence staining. For immunofluorescence assay, VM-ECs, HUVECs and HUSMCs were fixed with cold methanol (Fisher Chemicals) at 4 °C for 10 min and blocked in 5% horse serum (Vector Laboratories) in PBS. Anti-CD31 (1:50, Dako), anti-von Willebrand Factor (vWF) (1:100, Dako), anti-VE-Cadherin (1:50, Santa Cruz), anti-α-SMA (1:500, Sigma) and anti-PROX1 (1:50, R&D Systems) were incubated with primary antibody for 1 h followed by FITC which conjugated with secondary antibodies (1:200, Vector Laboratories). Nuclei were detected by DAPI counterstain (ProLong Gold Antifade Reagent with DAPI; Life Technologies). Fluorescent images were acquired using an immunofluorescence microscope (Nikon). Western blot assays were performed as previously described [24 (link)].
5
Immunofluorescent Analysis of NF-κB Translocation
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hMSCs were seeded onto coverslips in 4-well plates and cultured for one day. After LPS or inhibitor treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min and then permeabilized with cold methanol for 5 min. After blocking with 5% BSA in PBS for 1 hr, the cells were incubated with primary antibody overnight at 4 °C with anti-rabbit NF-κB p65 polyclonal antibody (1:200, Abcam, Cambridge, U.K) followed by incubation with an appropriate secondary donkey anti-rabbit IgG antibody (Jackson Laboratory, West Grove, PA) for 1 hr at room temperature. Following incubation, washing with PBS was performed three times and the cells were mounted with 4′, 6-diamidino-2-phenylindole (DAPI) containing mounting solutions (Vectashield, Vector Laboratories, Burlingame, CA) and imaged by immunofluorescence microscope (Nikon, Tokyo, Japan).
6
Visualizing Protein Localization in Live Cells
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pSDred-N1-VP7 and pEGFP-N1-HSP70 plasmids were transfected into CIK or 293T cells using Invigentech INVI DNA RNA, and cells expressing green or red (GFP or RFP) fluorescent fusion proteins were cultured for 36 h. After that, the cells were fixed with 4% paraformaldehyde for 10 min, stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, washed 3 times with phosphate-buffered saline (PBS), and treated with antifade mounting medium. Then the cells were analyzed with a immunofluorescence microscope (Nikon). For live-cell imaging, GCO cells were seeded on a chambered coverslip at a density of around 50% confluence. Cells were then transfected with corresponding plasmids (pEGFP-N1-HSP70, pDsRed-N1-VP7, pEGFP-N1, and pDsRed-N1) and pictures were recorded per 15 min using a Nikon confocal laser microscope system.
7
Immunofluorescence Analysis of RTA Protein
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iSLK/RTA-WT, iSLK/RTA-DD, SLK/iBAC.RTA-WT, or SLK/iBAC.RTA-Q37 cells were processed as previously described (53 (link)). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 buffer. After being blocked with goat serum, cells were incubated with primary mouse monoclonal anti-FLAG M2 antibody or polyclonal anti-RTA (1:100 dilution) serum. Cells were then incubated with Alexa Fluor 488–congugated goat secondary antibody (1:500 dilution), stained with 4′,6-diamidino-2-phenylindole (DAPI), and analyzed with confocal microscope (Nikon). Cells expressing fluorescent fusion proteins were fixed with 4% paraformaldehyde and directly analyzed by immunofluorescence microscope (Nikon). Representative images were shown for all analyses.
8
Exosome Isolation and Uptake Assay
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After tracheal intubation of the mice, the lungs were gently lavaged thrice with 1 mL of ice cold, sterile phosphate-buffered saline (PBS). The fluids collected from mice were maintained at −80°C and immediately processed for exosome isolation.
Exosome isolation was performed through differential ultracentrifugation as previously described (28 (link), 29 (link)). The concentration of protein in exosome pellets was measured using the bicinchoninic acid assay (Beyotime, Shanghai, China). For the in vitro uptake experiment, exosomes were labeled using a PKH67 green fluorescent cell linker mini kit (Sigma Aldrich, St. Louis, MO, USA) according to the instructions provided by the manufacturer. Ten random fields were counted. For the in vivo uptake study, the labeling procedure was identical to that used in the in vitro analysis. The labeled exosomes were delivered into the mice, and 18 h later the lungs were removed and embedded in OCT compound, and stored at −80°C. Photographs were captured using an immunofluorescence microscope (Nikon, Tokyo, Japan).
Exosome isolation was performed through differential ultracentrifugation as previously described (28 (link), 29 (link)). The concentration of protein in exosome pellets was measured using the bicinchoninic acid assay (Beyotime, Shanghai, China). For the in vitro uptake experiment, exosomes were labeled using a PKH67 green fluorescent cell linker mini kit (Sigma Aldrich, St. Louis, MO, USA) according to the instructions provided by the manufacturer. Ten random fields were counted. For the in vivo uptake study, the labeling procedure was identical to that used in the in vitro analysis. The labeled exosomes were delivered into the mice, and 18 h later the lungs were removed and embedded in OCT compound, and stored at −80°C. Photographs were captured using an immunofluorescence microscope (Nikon, Tokyo, Japan).
9
Immunofluorescence Imaging of Rat VSMCs
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Primary rat VSMCs cells (6 × 105 cells per well) were seeded in six-well plates with 12 % FBS at room temperature for 24 h. After washing in phosphate-buffered saline (PBS), cells were fixed for 30 min at 37° C in 4% paraformaldehyde, blocked with 3% bovine serum albumin (BSA) for 1 h, and incubated overnight at 4° C in PBS containing anti-rabbit α-SMA antibody (CST19245, 1:200). Subsequently, cells were washed three times with PBS and incubated for 45 min at 37° C with anti-rabbit secondary antibody Alexa Fluor 488 Donkey anti-Rabbit IgG (H+L) (34206ES60, 1:200). The images were acquired using an immunofluorescence microscope (Nikon, Tokyo, Japan) with ×40 magnification.
10
Exosome Uptake Assay in HK-2 Cells
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Exosomes derived from HK-2 cells treated with NG and HG for 48 h were isolated and identified as in our previous study (Tsai et al., 2020 (link)). In brief, cells were seeded at a density of 2 × 106 cells/100 mm dish and cultured for 48 h under NG or HG conditions in a cell culture medium containing 1% exosome-free serum (Life Technologies, Carlsbad, CA, USA). Exosomes were labeled with diluent C solution (20 μl, Life Technologies) and PKH26 (0.5 μl, Sigma-Aldrich Corp., St. Louis, MO, USA) for 5 min at room temperature and then incubated with HK-2 cells in the NG condition for 3 h. HK-2 cells were stained with Calcein-AM (1 μM, Life Technologies) for 30 min. The uptake of exosomes was found by using an immunofluorescence microscope (Nikon Instruments, Tokyo, Japan), and pictures were taken using a Nikon inverted fluorescence microscope (Eclipse TE200 microscope).
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