Primary MEFs were isolated from embryos as described previously (Tommasi et al. 2005 (link); Yoon et al. 2015 (link), 2019b (link)). Cells were pulse-labeled with 25 µM IdU (Sigma) for 20 min and subsequently washed twice with PBS buffer, followed by treatment with cisplatin (60 µM) and 250 µM CldU (Sigma) for 30 min. DNA fibers were spread on glass slides, and slides were incubated in 2.5 M HCl for 90 min and then washed with PBS buffer. The slides were incubated in blocking buffer (5% BSA in 1× PBS) for 2 h. Primary antibodies—rat anti-BrdU antibody (Abcam) and mouse anti-BrdU antibody (BD Bioscience)—were diluted in blocking buffer and incubated for 1 h followed by extensive washing with PBS buffer. Secondary antibodies—goat anti-rat Alexa 594 (Thermo Scientific) and goat anti-mouse Alexa 488 (Thermo Scientific)—were applied for 30 min and slides were mounted with antifade gold mounting medium (Invitrogen). DNA fibers were analyzed using a Nikon eclipse fluorescence microscope and quantified using NLS-Elements AR software.
Antifade gold mounting medium
Manufactured by Thermo Fisher Scientific
Antifade gold mounting medium is a laboratory reagent used to preserve and protect fluorescent signals in microscopy samples. It is designed to prevent the fading or quenching of fluorescent dyes, allowing for improved visualization and documentation of fluorescent-labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Hydrogen peroxide, by Thermo Fisher Scientific (2 mentions)
Dmem without glucose, by Thermo Fisher Scientific (2 mentions)
Bafilomycin, by Thermo Fisher Scientific (2 mentions)
Dmso cell culturegrade, by Thermo Fisher Scientific (2 mentions)
Antibodies for actin, by Cell Signaling Technology (2 mentions)
Vps34, by Cell Signaling Technology (2 mentions)
Ulk ser 317, by Cell Signaling Technology (2 mentions)
Ulk ser 757, by Cell Signaling Technology (2 mentions)
Atg13, by Cell Signaling Technology (2 mentions)
Atg 13pser 318, by Cell Signaling Technology (2 mentions)
Beclin ser 15, by Cell Signaling Technology (2 mentions)
Vps34 ser 249, by Cell Signaling Technology (2 mentions)
P ampk thr172, by Cell Signaling Technology (2 mentions)
6 protocols using antifade gold mounting medium
1
DNA Fiber Analysis of Replication and DNA Damage
2
Immunofluorescence Assay for Leishmania Cells
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The following protocol was carried out for immunofluorescence. Leishmania cells were washed twice with 1× PBS (phosphate buffer saline) and seeded in poly-lysine coated 10 well chamber slides or coverslips for 1 h to properly adhere cells at room temperature. The unadhered cells were washed with 1× PBS 4 to 5 times and fixed with 4% paraformaldehyde (Sigma) for 30 s and again washed with 1× PBS 4 to 5 times. The fixed cells were treated with 0.5% Triton X 100 (Sigma) for 15 min and subsequently washed, and incubated with 5% BSA (Sigma)in 1× PBS (blocking solution) for 1 h at room temperature. The blocking solution was removed and cells were incubated with the desired primary antibody for the incubation period as standardized for each antibody. This was followed by several washes and, incubation with required Alexa fluor 488 or Alexa 568 conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. The cells were washed and stained with nuclear dye 1× DRAQ5 (CST) for 10 min. The cells were washed 6 to 7 times to remove excess stain followed by drying and mounting with anti-fade gold mounting medium (Invitrogen). For curing the slides were kept 24 h at room temperature and high-resolution imaging was done in a confocal laser scanning microscope (Olympus FV3000). DNA–RNA hybrid and dsDNA were detected by S9.6 antibody (Merck) and dsDNA antibody (Abcam), respectively.
3
Autophagy Regulatory Pathway Analysis
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Hydrogen peroxide, bafilomycin, antifade gold mounting medium, FBS,
L-glutamine, penicillin /streptomycin, DMEM without glucose, DMEM without
sodium pyruvate for H2O2 treatment medium, PVDF membrane, DMSO cell culture
grade, Lipofectamine 2000, Lipofectamine 3000 were purchased from Thermo
Fisher Scientific. Polyfect was purchased from qiagen. Antibodies for actin,
LC3, ATG5, myc, VPS34, ULK ser 317, ULK ser 757, ATG 13, ATG 13pSer 318,
Beclin1, Beclin Ser 15, VPS34 Ser 249, AMPK, PAMPK Thr 172,Flag, GST,
HA,FIP200, ATG101 were purchased from Cell Signaling Technology. ULK1ser555
and ULK ser 777 were from Millipore. ULK1 for immunoprecipitation was
purchased from Sigma. ULK1 for western blots was purchased from Santacruz
Biotechnology. Anti-mouse IPMK was developed in our lab.
L-glutamine, penicillin /streptomycin, DMEM without glucose, DMEM without
sodium pyruvate for H2O2 treatment medium, PVDF membrane, DMSO cell culture
grade, Lipofectamine 2000, Lipofectamine 3000 were purchased from Thermo
Fisher Scientific. Polyfect was purchased from qiagen. Antibodies for actin,
LC3, ATG5, myc, VPS34, ULK ser 317, ULK ser 757, ATG 13, ATG 13pSer 318,
Beclin1, Beclin Ser 15, VPS34 Ser 249, AMPK, PAMPK Thr 172,Flag, GST,
HA,FIP200, ATG101 were purchased from Cell Signaling Technology. ULK1ser555
and ULK ser 777 were from Millipore. ULK1 for immunoprecipitation was
purchased from Sigma. ULK1 for western blots was purchased from Santacruz
Biotechnology. Anti-mouse IPMK was developed in our lab.
4
Autophagy Regulatory Pathway Analysis
5
Autophagy regulation by eNOS signaling
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Tunicamycin, chloroquine, N-acetyl cysteine (NAC), DETA-NONOate, mammalian cell lysis kit, Rabbit anti-actin antibody, DMEM:F12 medium, Mission esiRNA for SQSTM1, and eNOS siRNA were purchased from Sigma-Aldrich. Glycan Differentiation kit, WST-1 cell viability kit, and X-tremeGENE HP DNA Transfection reagent were obtained from Roche. ROS detection kit, gold antifade mounting medium, NuPAGE Novex 4-12% Bis-Tris gels, goat anti-rabbit IgG-alexa 488, Goat-anti-rabbit IgG-Alexa Fluor 568, First Strand cDNA synthesis kit, Fetal Bovine Serum were from Invitrogen. JC1 mitochondrial membrane potential assay kit was from Cayman. Apodirect TUNEL assay kit was purchased from Millipore. Ultralow attachment tissue culture plate was from Nunc. TurboFect siRNA Transfection Reagent, DAPI, and HRP chemiluminescence substrate were from Thermo Scientific. Anti-ubiquitin antibody (clone P4D1) was from Biolegend. Anti rabbit antibodies against LC3, p62, and calnexin, Rabbit mAb phosphor-p70 S6 Kinase (Thr 389), mouse anti-eNOS mAb were obtained from Cell signaling Technology. pcDNA3-eNOS-GFP was obtained from Addgene. LC3-GFP and p62-EGFP constructs were kind gifts from Dr. Beth Levine (Columbia University, NY) and Dr. Terje Johansen (University of Tromso, Norway), respectively.
6
SINE Compound Immunocytochemistry Protocol
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Cells were seeded on 4 well glass chambered slides (Corning, USA) and exposed to SINE compounds at indicated doses for 24 hrs. After the treatment period was over, the cells were fixed using 4% paraformaldehyde solution for 10 min followed by permeabilization using 0.5% Triton (Sigma-Aldrich, USA). The cells were washed with PBS followed by blocking in 2% BSA solution in TBST for 30 minutes. The blocked slides probed with different primary antibodies overnight at 1:100 dilution and appropriate Alexa Fluor conjugated secondary antibody (1:100 dilution) for 2 hrs. The slides were air dried and mounted using the mounting medium (Gold Antifade mounting medium Invitrogen) and covered with a coverslip and sealed with nail polish. The slides were analyzed under a fluorescent microscope at 40× magnification (Evos FL microscope system).
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