Mesangial cells were seeded onto round glass dishes and then subjected to various treatments. After they were washed three times with cold PBS, the cells were fixed with cold methanol at − 20 °C for 15 min and then incubated in blocking buffer (1% BSA and 0.1% Triton X-100 in PBS, pH 7.4) for 30 min at room temperature. The cells were subsequently incubated with Nrf2 (1:200), Keap1 (1:100), collagen IV (1:200), and laminin (1:100) primary antibodies for 12 h at room temperature. Then, the cells were washed three times with PBS and incubated with a secondary antibody conjugated to DyLight 594 or DyLight 488 (Earthox, Millbrae, CA, USA) at 37 °C for 1 h in the dark. The cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 1 min. Coverslips were mounted onto the glass slides, and the cells were viewed with an Olympus BX43F fluorescence microscope (Tokyo, Japan).
Dylight 594
Manufactured by EarthOx
Sourced in United States
DyLight 594 is a fluorescent dye used in laboratory applications. It has an excitation wavelength of 593 nm and an emission wavelength of 614 nm. The dye is designed for use in various biological assays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 488, by EarthOx (3 mentions)
Bx43f fluorescence microscope, by Olympus (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lc3a b, by Affinity Biosciences (1 mentions)
Carm1, by Affinity Biosciences (1 mentions)
H3r17me3a, by Affinity Biosciences (1 mentions)
E032420, by EarthOx (1 mentions)
Dylight 488, by Abbkine (1 mentions)
Eclipse ti s, by Nikon (1 mentions)
Anti ripk3, by Santa Cruz Biotechnology (1 mentions)
Anti ripk1, by Cell Signaling Technology (1 mentions)
Anti nrf2 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
6 protocols using dylight 594
1
Immunofluorescence Analysis of Mesangial Cells
2
Immunofluorescence Analysis of Cellular Proteins
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Cells were seeded on slide covers in 24‐well plates, grown to 80% confluence, and treated as described in 2.10. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS three times (10 minutes each), permeabilized with 0.25%Triton X‐100 in 3% BSA for 10 minutes, washed with PBS three times, then blocked in 5% BSA at room temperature for 40 minutes. After washing with PBS, the cells were incubated with antibodies against LC3A/B (1:250, Affinity), AMPK (1:200, Affinity), SKP2 (1:200, Affinity), CARM1 (1:250, Affinity) and H3R17me3a (1:200, Affinity) at 4°C overnight. Then, the cells were washed with PBS and incubated with a fluorescent secondary antibody (DyLight 594, E032420, EarthOx, USA; DyLight 488, A23220, Abbkine) in the dark for 60 minutes, stained with DAPI for 10 minutes, then washed twice with PBS. Images were captured using a fluorescence microscope (Eclipse Ti‐S, Nikon, Japan).
3
Immunofluorescence Staining of RIPK1 and RIPK3
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The cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The cells were blocked with 3% BSA (Sigma-Aldrich, St Louis, MO, USA) at room temperature for 30 minutes. Next, the cells were incubated with anti-RIPK1 (Cell Signaling Technology, Inc., USA) and anti-RIPK3 (Santa Cruz Biotechnology, CA) antibodies for 2 hours at 37 °C. Then, the cells were rinsed with PBS and incubated with Dylight 594 and DyLight 488 AffiniPure secondary antibodies (EarthOx, San Francisco, CA, USA) for 1 hour. Next, the cells were counterstained with DAPI for 15 minutes at 37 °C in the dark. After being washed with PBS, the cells were visualised by confocal microscopy (Carl Zeiss LSM700).
4
Nrf2 Immunofluorescence Staining and Imaging
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Cells plated on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, treated with blocking medium (1% bovine serum albumin in PBS) for 10 min, and then incubated with an anti-Nrf2 antibody (Abcam, Cambridge, UK) at 4°C overnight. The immune-reacted primary Nrf2 antibody was detected following a 1-h incubation in the dark at 37°C with a secondary antibody conjugated with DyLight 594 (EarthOx, Millbrae, CA, USA). The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA, USA) for 2 min in the dark at room temperature and washed. Then, the cells were mounted onto microscope slides in mounting medium. Observations were carried out using an Olympus BX43F fluorescence microscope.
5
Immunofluorescent Labeling of Cells
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Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C after blocking with 10% goat serum. After washing with 1 × PBS, the cells were incubated with secondary antibodies conjugated with DyLight 488 or DyLight 594 (1:100) (Earthox, Millbrae, CA, USA) for 1 h at 37 °C. The cells were stained with DAPI (Beyotime, Nantong, China) to visualize the nuclei.
6
Immunocytochemistry of Differentiated Podocytes
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Differentiated podocytes were fixed with cold methanol at −20°C for 20 min and permeabilized with 0.1% Triton X-100/PBS. After they were washed three times with cold PBS. The cells were blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature and incubated with mouse anti-synaptopodin antibody (1:200, santa cruz biotechnology, sc-515842) or rabbit anti-cleaved caspase three antibody (1:400 Cell Signaling Technology, 9661) at 37°C for 2 h or 4°C overnight. Then, the cells were washed three times with PBS and incubated with a secondary antibody conjugated to DyLight 488 or DyLight 594 (Earthox, Millbrae, CA, United States) at 37°C for 1 h, respectively. Nuclei were counterstained with DAPI. The coverslips were mounted onto glass slides, and the images were viewed with an Olympus BX43F fluorescence microscope (OLYMPUS, Japan).
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