Cells were cultured on coverslips and fixed with 4% paraformaldehyde/PBS, and then they were incubated overnight with anti‐HSP60 (1:300; Santa Cruz Biotechnology) or anti‐LC3B (1:250; Cell Signaling Technology) antibodies at 4°C in a darkroom. After washing and incubating with a fluorescently labeled secondary antibody, either IgG‐Alexa Fluor 594 or IgG‐Alexa Fluor 488 (1:300; Cell Signaling Technology). A final incubation was performed to stain the cells with DAPI (Beyotime)(Zhou et al., 2018 ). Images were captured using a confocal laser microscope at 600 × magnification (Nikon). Pearson's correlation coefficients were calculated with Image J v2.4.1.7 using 40 cells from 10 images (four cells per image) for each cell line (Zinchuk et al., 2013 ). Fragmented mitochondria‐containing cells were quantitatively defined using Image J, according to published criteria (Wang et al., 2016 ).
Igg alexa fluor 594
Manufactured by Cell Signaling Technology
Sourced in United States
IgG‐Alexa Fluor 594 is a fluorescently labeled secondary antibody. It is designed to detect and visualize primary antibodies in various immunoassays. The Alexa Fluor 594 dye provides strong, red-orange fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Anti hsp60, by Santa Cruz Biotechnology (2 mentions)
Igg alexa fluor 488, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Laser confocal microscope, by Nikon (1 mentions)
Anti phospho erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Ap53886pu n, by OriGene (1 mentions)
Af1009, by Affinity Biosciences (1 mentions)
Sw3015, by Solarbio (1 mentions)
4 protocols using igg alexa fluor 594
1
Automated Mitochondrial Morphology Analysis
2
Multimodal Mitochondrial Imaging and Protein Localization
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Cells were cultured on coverslips and incubated in DMEM containing 100 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C in the dark. After staining, cells were washed with fresh growth medium, and fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 min. Cells were then permeabilized with PBS containing 0.2% Triton X-100 for 3 min at room temperature, and incubated overnight with anti-HSP60 (1:300; Santa Cruz Biotechnology) or anti-phospho-ERK (Thr202/Tyr204) (1:250; Cell Signaling Technology) antibodies at 4 °C in a darkroom. Next, cells were washed with PBS and incubated with a fluorescently labeled secondary antibody either IgG-Alexa Fluor 594 or IgG-Alexa Fluor 488 (1:300; Cell Signaling Technology) for 1 h at room temperature in the dark. A final incubation was performed to stain the cells with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Jiangsu, China) for 15 min at room temperature. After mounting the coverslips, images were captured using a confocal laser microscope at a magnification of ×600 (Nikon, Telford, UK).
3
Immunofluorescence Staining Protocol
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Triton X-100 (0.5%, Sigma-Aldrich, St. Louis, MO, USA) was used to increased cytomembrane permeability. After being blocked with blocking buffer (1% bovine serum albumin, 22.52 mg/mL glycine, and 0.1% Tween-20 in phosphate-buffered saline) for 30 min, the membrane was incubated with primary antibody dilution buffer, IgG-Alexa Fluor® 594 (Cell Signaling Technology, Beverly, USA), DAPI (Solarbio), and antifade mounting medium (Thermo Fisher Scientific, Waltham, MA, USA), successively. The membranes were kept in the dark throughout the process.
4
Immunofluorescence Analysis of Lung AT2 Cells
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Lung tissue was fixed with 4% PFA for at least 48 h, and then dehydrated and embedded in paraffin. The embedded lung tissue was sectioned into slices with a thickness of 4 μm and subsequently affixed onto slides for staining. AT2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.5% Tryon-100 for 30 min. Cells washed with PBS were blocked with 5% bovine serum albumin (BSA) (SW3015, Solarbio, Beijing, China) for 1 h at room temperature.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.
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