Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline. Permeabilization and staining with anti-AIF (sc-13116, SantaCruz Biotechnology), anti-γH2AX (Ab 22551, Abcam), anti-RPA70 (clone 2H10, Sigma-Aldrich) was performed as described [28] (link). Cells were counterstained with DAPI before microscopic examination using 60× magnification and a Nikon Eclipse 80i microscope. Images were acquired using Genikon (Nikon) software and processed with Adobe Photoshop.
Sc 13116
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-13116 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for scientific research purposes. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Genikon, by Nikon (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Ab22551, by Abcam (1 mentions)
Alexa fluor 488 af488, by Thermo Fisher Scientific (1 mentions)
A5316, by Merck Group (1 mentions)
Mouse monoclonal antibody against cox 4, by Abcam (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Ab39165, by Abcam (1 mentions)
Alexa conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Plan apochromat objective, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by PerkinElmer (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
A0216, by Beyotime (1 mentions)
6 protocols using sc 13116
1
Immunofluorescence Assay for DNA Damage
2
Immunofluorescence and Western Blot Protocols
3
Immunostaining of Retinal Sections
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Cell cultures on the coverslips were fixed for 20 min with 4% PF and the rat retinal sections were recovered to RT. The coverslips or retinal sections were washed three times for 5 min in ice-cold 0.01 M PBS. Subsequently, the coverslips or retinal sections were blocked for 1 h in blocking buffer, i.e., PBS containing 5% normal bovine serum and 0.3% Triton X-100, and then incubated with combinations of the primary antibodies against the following targets, overnight at 4°C: tAIF (1:500, sc-13116, Santa Cruz), CAST (1:50, sc-2-7799, Santa Cruz, Dallas, TX, United States) and calpain2 (1:100, ab39165, Abcam, Cambridge, United Kingdom). The coverslips or retinal sections were shifted to RT for 30 min, washed three times as described above, and incubated with Alexa-conjugated secondary antibodies (1:200, Jackson Immuno Research, West Grove, PA, United States) for 2 h. After washing three times in PBS, the coverslips or retinal sections were covered with Vectashield mounting medium containing DAPI. The coverslips or retinal sections were stained in parallel and images were acquired, with a fluorescence microscope, using the same settings.
4
Immunofluorescence Imaging of AIF in LoVo Cells
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A density of 75 × 103 LoVo cells were seeded on coverslips placed in the bottom of each well in 12 well plates. 48 h after inhibition by siRNAs, cells were fixed in 4% PFA solution for 30 min at RT and then washed with washing buffer. Non-specific binding was blocked using block solution containing 10% FBS. Cells were incubated in anti-AIF prepared in block solution (1:250; sc-13,116; Santa Cruz) for 30 min at RT. Then washed two times with washing buffer, followed by 30 min incubation in secondary Alexa fluor 488 donkey anti-mouse IgG (A-21202, Thermo Fisher). Coverslips were mounted on slides using mounting solution. All images were captured using LSM 800 (Zeiss, Jena, Germany) inverted confocal microscope equipped with a 63x Plan-Apochromat objective (NA1.4 oil).
5
Western Blot Analysis of AIF Protein
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Cells were harvested and lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate (Sigma), 0.1% SDS (Sigma), 50 mM Tris–HCl (pH 7.4), 150 mM NaCl (Sigma)) containing protease Inhibitor Cocktail (Roche Applied Science) for 15 min on ice. Cell lysate was centrifuged at 16000×g at 4 °C for 15 min and protein concentration was measured by Pierce BCA Protein assay kit (ThermoFisher). Equal amounts of proteins were loaded on a 10% SDS-PAG and after separation by electrophoresis, transferred onto nitrocellulose membranes (Bio-Rad). Membranes were then blocked in 5% non-fat milk for 1 h at RT and immunoblotted by overnight incubation at 4 °C with the indicated anti-AIF primary antibody (1:1000; sc-13,116; Santa Cruz). After three washes with PBS-Tween (0.1%), the membranes were incubated with anti-mouse secondary antibody: anti-IgG (1:2000; sc-2025; Cell Signaling) for 1 h at RT. Finally, the protein bands were visualized using an Enhanced Chemiluminescence Detection kit (Perkin Elmer, Waltham, MA, USA).
6
Immunofluorescence and Western Blot Analysis
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The following antibodies were used: anti-AIF (ab32516, Abcam, USA; 1:300 for IF), anti-AIF (sc-13 116, Santa Cruz, USA; 1:1000 for WB), anti-CtBP2 (612044, BD, USA; 1:200 for IF), anti-γH2AX (JBW301, Merck, Germany; 1:300 for IF), anti-β3-Tubulin (MA1-118, Invitrogen, USA; 1:300 for IF), anti-β-actin (AF0003, Beyotime, China; 1:1000 for WB), Alexa Fluor 555-Phalloidin (A34055, Invitrogen; 1:1000 for IF), Alexa Fluor 488, 555, and 647 secondary antibodies (A-11008, A-11001, A-31572, A-31571, Invitrogen; 1:500 for IF), and anti-mouse horse radish peroxidase (HRP)-conjugated secondary antibodies (A0216, Beyotime; 1:5000 for WB). The reagents were: phosphate buffered saline (PBS) (P1010, Solarbio, China), 10% EDTA decalcifying solution (E1171, Solarbio), 4% paraformaldehyde (PFA) fixing solution (P0099, Beyotime), RIPA lysis buffer (P0013B, Beyotime), phenylmethanesulfonyl fluoride (PMSF) (36978, Thermo Fisher, USA), optimal cutting temperature compound (OCT) (4583, SAKURA, USA), Dulbecco’s modified Eagle medium-high glucose (DMEM-HG) (11965092, Gibco, USA), fetal bovine serum (FBS) (10099141C, Gibco), penicillin-streptomycin (15140122, Gibco), and DAPI (ZLI-9557, ZSGB-BIO, China).
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