The expression of cytochrome c was analyzed by immunofluorescence staining. Cells grown on glass-bottomed dishes (MatTek, Ashland, MA, USA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS) for 10 min. Slides were air-dried, washed with PBS, and incubated with anti-cytochrome c (1:25; Abcam, Cambridge, UK) in 3% bovine serum albumin in PBS. Further details on the protocol can be found in our previous article [68 (link)]. Images were observed under a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were analyzed using Zeiss LSM Image Browser, version 4.2.0121.
Formaldehyde solution
Manufactured by R&D Systems
Sourced in United States, United Kingdom
4% formaldehyde solution is a laboratory reagent used for fixation and preservation of biological samples. It is a clear, aqueous solution containing 4% formaldehyde. This solution is commonly used in various biological and histological applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm image browser, by Zeiss (5 mentions)
Lsm 700 meta, by Zeiss (5 mentions)
Glass bottomed dishes, by MatTek (4 mentions)
Alexa 488, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Anti cytochrome c, by Abcam (2 mentions)
Anti β catenin, by Abcam (2 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
5 protocols using formaldehyde solution
1
Cytochrome c Immunofluorescence Staining
2
Immunofluorescence Analysis of β-Catenin
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The expression of β-catenin was analyzed by immunofluorescence staining. Cells grown on glass-bottomed dishes (MatTek, Ashland, MA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 minutes and permeabilized with 0.5% TritonX-100 in phosphate buffered saline (PBS) for 10 minutes. Slides were air-dried, washed with PBS, and incubated with anti-β-catenin (1:25; Abcam, Cambridge, UK) in 3% bovine serum albumin in PBS. After being washed with PBS, slides were incubated with Alexa 488 (1:200; Molecular Probes, Eugene, OR) Nuclei were stained with Hoechst 33,342 (Life Technologies, Grand Island, NY) for visualization. Images were observed under a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were analyzed using Zeiss LSM Image Browser, version 4.2.0121.
3
Immunofluorescence Analysis of β-Catenin
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The expression of β-catenin was analyzed by immunofluorescence staining. Cells cultured on glass-bottomed dishes (MatTek, Ashland, MA. USA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS) for 10 min. Slides were air dried, washed with PBS, and incubated with anti-β-catenin (1:25; Abcam, Cambridge, UK) in 3% bovine serum albumin in PBS. After being washed with PBS, slides were incubated with Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA). Nuclei were stained with Hoechst 33342 (Life Technologies, Grand Island, NY, USA) for visualization. Images were observed under a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were analyzed using the Zeiss LSM Image Browser, version 4.2.0121.
4
Quantifying PMCA and PGC1α Expression
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The expression levels of PMCA and PGC1α were analyzed by immunofluorescence staining. Cells cultured on glass-bottomed dishes (MatTek, Ashland, MA, USA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. The slides were air dried, washed with PBS, and incubated overnight at 4 °C with anti-PMCA (1:50, Abcam, #2825) and anti-PGC1α (1:25; Abcam, Cambridge, UK, #106814) in 3% bovine serum albumin which included PBS. After being washed with PBS, slides were incubated with Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA) for 1–2 h at room temperature. Nuclei were stained with Hoechst 33342 (Life Technologies) for visualization. Images were observed under a confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and analyzed using the Zeiss LSM Image Browser, version 4.2.0121.
5
Cytochrome c Localization Assay
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Cytochrome c release from the mitochondria was analyzed using immunofluorescence staining. Cells were grown in glass-bottom dishes (MatTek, Ashland, MA), fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 minutes, and then permeabilized with 0.5% Triton X-100 (in PBS) for 10 minutes. The slides were air-dried, washed with PBS, and incubated with anti–cytochrome c (1:25; Abcam, Cambridge, UK) in 3% bovine serum albumin in PBS. After washing with PBS, the slides were incubated with Alexa 488 (1:200; Molecular Probes, Eugene, OR), and the nuclei were stained with Hoechst 33342 (Life Technologies, Grand Island, NY) for visualization. The images were observed under a confocal microscope (LSM Meta 700, Zeiss, Oberkochen, Germany) and were analyzed using the Zeiss LSM image browser, version 4.2.0121.
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