RVECs seeded in 24-well plates were pretreated with/without HCQ for 10 h and incubated with TNF-α for another 24 h at 80%–90% confluency. After stimulation, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% BSA for 2 h at room temperature. Subsequently, they were stained overnight with rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C, washed with PBS, incubated with Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, California, USA) for 2 h, and then with DAPI (Abcam, Cambridge, UK) for 10 min. Cells were imaged using an inverted fluorescent microscope (Nikon).
Inverted fluorescent microscope
Manufactured by Nikon
Sourced in Japan, United States
The Inverted Fluorescent Microscope is a specialized optical instrument designed to observe and analyze fluorescently labeled samples. It features an inverted configuration, where the light source and objectives are positioned below the stage, allowing for the observation of cells and tissues cultured on coverslips or multi-well plates from the underside. This microscope is commonly used in various fields, including cell biology, molecular biology, and biomedical research, to visualize and study fluorescently tagged proteins, organelles, and other cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Ri1 color cooled camera system, by Nikon (2 mentions)
Glass coverslip bottomed culture dishes, by MatTek (2 mentions)
Dq gelatin fitc, by Thermo Fisher Scientific (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Oil red o working solution, by Solarbio (2 mentions)
Acridine orange, by Merck Group (2 mentions)
Nis advanced research software, by Nikon (2 mentions)
Basic research software, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Nis elements advanced research software, by Nikon (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Dihydroethidium, by Beyotime (2 mentions)
Rabbit anti phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
Alexa 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Nis elements ar imaging software, by Nikon (1 mentions)
Lsm710 confocal microscope, by Nikon (1 mentions)
83 protocols using inverted fluorescent microscope
1
Investigating NF-κB Activation in RVECs
2
Quantifying Neuroinflammatory Markers in Hippocampus
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Images were obtained using a Nikon Inverted Fluorescent Microscope (Nikon, Tokyo, Japan). The number of CD11b-, Iba-1-, CD68-, GFAP-, and Nissl-positive cells were quantified in the CA1 and CA3 subregions of the dorsal hippocampus (−1.46 and −1.70 mm from bregma) using the NIS elements AR imaging software (Nikon, Tokyo, Japan).
3
Immunofluorescence Staining of Cultured Scaffolds
4
Immunofluorescence Cell Imaging Protocol
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The cells were cultured in glass coverslip-bottomed culture dishes (MatTek, Ashland, MA) to 50–80% confluence. After the culture medium was aspirated, the cells were rinsed with PBS twice, fixed with 3.7% paraformaldehyde at 25 °C for 10 min, and permeabilized with 0.2% Triton X-100 in PBS at 25 °C for 10 min. After washing with PBS, the cells were incubated with primary antibody at 8 °C overnight. The cells were washed with PBS three times and incubated with a fluorescent dye-conjugated secondary antibody and phalloidin with at 37 °C for 1–2 h. After washed with PBS three times, fluorescent staining of the cells were visualized under Zeiss LSM710 confocal microscope or Nikon inverted fluorescent microscope.
5
Lentiviral Transduction of hFL-MSCs
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Cultured 70–80% confluent hFL-MSCs were exposed to green fluorescent protein (GFP)-encoding lentiviral vector (pLVIRES-GFP). The cells were transduced with pLVIRES-GFP at the multiplicity of infection in the presence of 5 mg/ml polybrene and the second transduction was repeated after 48 h. Subsequently, transduced cells were evaluated for expression of GFP using inverted fluorescent microscope (Nikon, Japan) (27 (link)).
6
Immunofluorescence Staining Protocol
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The cells were cultured in glass bottom microwell dishes (MatTek) to 50–80 % confluence. After the culture medium was removed, the cells were rinsed with PBS twice, fixed with 3.7 % paraformaldehyde at 25 °C for 10 min and permeablized with 0.2 % Triton X-100 in PBS at 25 °C for 10 min. Following washing with PBS, the cells were incubated with primary antibody at 25 °C for 60 min. Then the cells were washed with PBS three times and incubated with secondary antibody that was conjugated with a fluorescent dye at 25 °C for 60 min. Finally, the cells were washed with PBS three times (for 10 min each) and incubated in PBS. Immunofluorescence staining was visualized with a Nikon inverted fluorescent microscope.
7
Cell Viability and Colony Formation
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Cells were harvested by trypsinization, and viability assessed by trypan blue exclusion under a phase-contrast microscope. Equal number of cells were seeded. Cell numbers were determined by hemocytometric counting for 5 consecutive days. For the soft agar assay, cells were treated with doxycycline (2 μg/ml) for 24 h, and mixed with 0.33% agarose in DMEM and overlaid on 0.5% agarose in a 12-well plate. After 4 weeks, colony formation was examined by staining colonies with 0.005% crystal violet (Sigma-aldrich) and stained colonies were counted using an inverted fluorescent microscope (Nikon, Seoul, Rep. of Korea).
8
Apoptosis Assessment of MCTS Cultures
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Apoptosis assessment of MCTS cultures using acridine orange and propidium iodide double staining was carried out according to the method described previously [14 ]. The staining dye (10 μg/ml of acridine orange and propidium iodide each) was added into MCTS cultures and 10 μl of the stained cells were observed under an inverted fluorescent microscope (Nikon, Japan).
9
Immunofluorescent Characterization of Cells
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Fresh cells were seeded at a suitable density on microscope slides, washed three
times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized by
treating with 0.5% Triton X-100 for another 10 min. The cells were blocked by
treating with 3% bovine serum albumin (BBI, Shanghai, China) for 1 h. Rat
anti-vimentin (1:100; Cell Signaling Technologies, Danvers, MA, USA) and rabbit
anti-CD34 (1:200; Abcam, Cambridge, UK) antibodies were used. The cells were
treated with the antibodies overnight at 4°C. After washing three times with
PBS, the cells were treated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488
(1:1000; Abcam, Cambridge, UK) or goat anti-mouse IgG (H+L) Alexa Fluor 568
(1:1000; Abcam, Cambridge, UK) at 37°C for 1 h, followed by treatment with
4’,6-diamidino-2-phenylindole (DAPI; Cayman Chemical, Ann Arbor, MI, USA). The
slides were fixed in an antifade medium (1:1000; Beyotime, Shanghai, China) and
imaged using an inverted fluorescent microscope (Nikon, Tokyo, Japan).
times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized by
treating with 0.5% Triton X-100 for another 10 min. The cells were blocked by
treating with 3% bovine serum albumin (BBI, Shanghai, China) for 1 h. Rat
anti-vimentin (1:100; Cell Signaling Technologies, Danvers, MA, USA) and rabbit
anti-CD34 (1:200; Abcam, Cambridge, UK) antibodies were used. The cells were
treated with the antibodies overnight at 4°C. After washing three times with
PBS, the cells were treated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488
(1:1000; Abcam, Cambridge, UK) or goat anti-mouse IgG (H+L) Alexa Fluor 568
(1:1000; Abcam, Cambridge, UK) at 37°C for 1 h, followed by treatment with
4’,6-diamidino-2-phenylindole (DAPI; Cayman Chemical, Ann Arbor, MI, USA). The
slides were fixed in an antifade medium (1:1000; Beyotime, Shanghai, China) and
imaged using an inverted fluorescent microscope (Nikon, Tokyo, Japan).
10
Characterizing Endothelial Progenitor Cells
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After 7 day in culture, attached EPCs were labeled with Dil-acLDL (10 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) and FITC-labeled UEA-1 lectin (10 µg/ml; Sigma-Aldrich; Merck KGaA) for 1 h. After washing with PBS three times, cells were observed under an inverted fluorescent microscope (Nikon Corporation). Cells demonstrating double-positive fluorescence of Dil-acLDL and lectin were identified as differentiating EPCs. EPCs from one mouse were used for fluorescence staining.
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