Protein quantitation was performed by using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Mass). Anti-p62 was purchased from Santa Cruz Biotechnology (Dallas, Tex). Anti-LC3 antibodies were purchased from Cell Signaling Technology (Danvers, Mass). These experiments were performed by T.P.F.G., M.S.N., and W.W., who had 7 years of experience in cancer research.
Anti lc3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
The Anti-LC3 antibody is a laboratory reagent used to detect the protein LC3 (Microtubule-associated proteins 1A/1B light chain 3B) in cellular samples. LC3 is a key marker for autophagy, a process of cellular degradation and recycling. The antibody can be used in various immunodetection techniques, such as Western blotting, immunocytochemistry, and immunohistochemistry, to visualize and quantify the expression of LC3 in biological samples.
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Lab products found in correlation
Lsm 880, by Zeiss (3 mentions)
Anti p62 antibody, by Cell Signaling Technology (3 mentions)
Anti rabbit hrp linked secondary antibody 7074p2, by Merck Group (2 mentions)
Goat anti mouse hrp linked secondary antibody 62 6520, by Merck Group (2 mentions)
Anti total s6, by Cell Signaling Technology (2 mentions)
Anti phospho s6, by Cell Signaling Technology (2 mentions)
Anti p62, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Anti sqstm1 p62 antibody, by Abcam (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Keratinocyte sfm 1 medium, by Thermo Fisher Scientific (1 mentions)
Hk 2 cells, by Thermo Fisher Scientific (1 mentions)
Hk 2 cell, by American Type Culture Collection (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
49 protocols using anti lc3 antibody
1
Quantifying Protein Levels in Cancer Research
2
Autophagy Flux Assay in HK-2 Cells
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HK-2 cells, which are human kidney proximal tubular cells, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HK-2 cells were maintained in Keratinocyte-SFM (1×) medium (Life Technologies Green Island NY).
Autophagic flux was determined by analyzing LC3-II turnover by preventing lysosomal degradation using CQ (Sigma-Aldrich, St. Louis, MO, USA). LC3-II expression was evaluated by western blotting using anti-LC3 antibodies (Cell Signaling Technology, Danvers, MA, USA) [37 (link), 46 (link)]. LC3-flux was calculated by subtracting the densitometry value of normalized LC3-II in the sample treated with CQ by the value in the sample treated without CQ [37 (link), 46 (link)]. LC3-II flux was expressed relative to the respective controls [37 (link), 46 (link)]. HK-2 cells were cultured in Hanks’ Balanced Salt Solution (HBSS), amino acid-free solution, with SAM (0.5 mM, 2 mM or 4 mM) or vehicle for 2 hours, and then 50 μM CQ or vehicle was administered for 1 hour before collection of samples.
Autophagic flux was determined by analyzing LC3-II turnover by preventing lysosomal degradation using CQ (Sigma-Aldrich, St. Louis, MO, USA). LC3-II expression was evaluated by western blotting using anti-LC3 antibodies (Cell Signaling Technology, Danvers, MA, USA) [37 (link), 46 (link)]. LC3-flux was calculated by subtracting the densitometry value of normalized LC3-II in the sample treated with CQ by the value in the sample treated without CQ [37 (link), 46 (link)]. LC3-II flux was expressed relative to the respective controls [37 (link), 46 (link)]. HK-2 cells were cultured in Hanks’ Balanced Salt Solution (HBSS), amino acid-free solution, with SAM (0.5 mM, 2 mM or 4 mM) or vehicle for 2 hours, and then 50 μM CQ or vehicle was administered for 1 hour before collection of samples.
3
Immunofluorescent Analysis of Autophagy Markers
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Lung tissue paraffin Sects. (4 μm thick) were dewaxed and hydrated. Antigen retrieval was then performed by microwaving the sections in sodium citrate buffer (pH 6.0) for 15 min and cooling to room temperature. Then, 0.3% hydrogen peroxide was used to block endogenous peroxidase activity in the sections, followed by PBS washing for 15 min. Subsequently, 5% goat serum was used to block the samples for 1 h at room temperature. After discarding the goat serum, the samples were incubated overnight at 4 °C with anti-LC3 antibodies (Cell Signaling Technology) and anti-RAB7 antibodies (Sigma-Aldrich). After being returned to room temperature, the sections were PBS-washed. Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L (Abcam) and Alexa Fluor 555-conjugated donkey Anti-Mouse IgG H&L (Abcam) were then incubated with a portion of the sections, followed by PBS washing, DAPI staining, and observation under a laser confocal microscope (Nikon A1R, Tokyo, Japan).
4
Induction of Autophagy in Mesangial Cells
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Hemin, zinc protoporphyrin (ZnPP), and curcumin were purchased from Sigma-Aldrich Co. (USA). The anti-LC3 antibodies were obtained from Cell Signaling Technology, Inc. (USA). A stable human mesangial cell line (kindly donated by Dr. J. D. Sraer, Hopital Tenon, Paris, France) was established by transfection and immortalization by the viral oncogene large T-SV40 of human mesangial cells isolated from normal human glomeruli [16] (link), and were cultured as described previously [17] (link). In brief, these cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The cells between passages 3 and 15 were used and then sub-cultured in serum-deprived medium (1% FBS) for 12 h before use in subsequent experiments. The cells were treated with 125 µM H2O2 for 24 h.
5
Autophagy Modulation in Neurodegeneration
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Oxyresveratrol, 3-methyladenine (3-MA), rapamycin, chloroquine diphosphate salt (CQ), and dexamethasone (DEX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-LC3 (D3U4C) XP Rabbit mAb (Alexa Fluor 488 Conjugate), anti-SQSTM1/p62, anti-phospho-S6, anti-total S6, anti-LAMP1, APP, and anti-LC3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MAP2 (microtubule-associated protein 2) antibody (ab5392), goat anti-chicken IgY H&L (Alexa Fluor 647) (ab150171), and corticosterone (ab14597) were obtained from Abcam (Cambridge, UK). Anti-rabbit HRP-linked secondary antibody (#7074P2) and goat anti-mouse HRP-linked secondary antibody (#62-6520) were purchased from Sigma-Aldrich and Invitrogen (Carlsbad, CA, USA), respectively. ULK1 siRNA (m): (sc-44849) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). OxyR, CQ, rapamycin, and compound C were dissolved in dimethyl sulfoxide and 3-MA was dissolved in double-distilled water.
6
FT895 Induced Autophagy Visualization
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Cells were grown on Ibid tissue culture dishes (IB-81156, Ibidi, Gräfelfing, Germany) overnight and were treated with FT895 for 24 h. Treated cells were washed with PBS twice and fixed in 4% paraformaldehyde at room temperature (RT) for one minute, followed by fixing with cold methanol at −20 °C for 20 min. The fixed cells were blocked with PBS containing 5% fetal bovine serum for one hour at RT. For immunostaining, the primary antibodies, anti-ATP5H (Thermo Fisher Scientific, Waltham, MA, USA), and anti-LC3 antibodies (Cell Signaling, Danvers, MA, USA), were added into the wells and incubated overnight at 4 °C. The cell nuclei were stained with DAPI for 10 min at RT, and the cells were mounted using a mounting medium for microscopy. The confocal images were acquired with a Zeiss LSM 880 (Zeiss, Oberkochen, Germany).
7
Immunofluorescence Analysis of Autophagy
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The PROM1/CD133+ and PROM1/CD133− cells were plated on Tissue Culture Treated Glass Slide (BD Falcon, 354114) and subjected to PDT treatment (1.3 J/cm2). After 24 h, cells were washed with PBS and fixed with 3.7% paraformaldehyde for 8 min. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, blocked with 3% BSA for 60 min, and incubated with anti-LC3 antibodies (1:200) (Cell Signaling Technology, 3868S) overnight. After washing with PBS, cells were incubated for 1 h with DyLight 488 conjugated secondary antibody (1:500) (BioLink Biotechnology, B0210). Nuclei were counterstained with DAPI (Biotium, 40043) for 5 min. Slides were mounted with a coverslip using Fluoro-Gel (Electron Microscopy Sciences, 17985-10). Cells were imaged using a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany), and the images were analyzed using a Leica Application Suite 2.02. DyLight 488 was excited using an argon laser (488 nm), and DAPI was excited using a diode laser (405 nm). The emission lights for DAPI and DyLight 488 were collected between 410 and 490 nm and between 495 and 540 nm, respectively.
8
Autophagy Modulation in Cell Experiments
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BXZ2, 3-methyladenine (3-MA), rapamycin, chloroquine diphosphate salt (CQ), alloxan monohydrate, and OSMI-1 were purchased from Sigma-Aldrich (St. Louis, MO 63178, USA). Anti-LC3 (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate), anti-SQSTM1/p62, anti-phospho-S6, anti-total S6, anti-LAMP1, and anti-LC3 antibodies were purchased from Cell Signaling Technology (Danvers, MA 01923, USA). O-linked-N-acetylglucosamine antibody (RL2) was purchased from Thermo Fisher (Rockford, IL 61101, USA). Anti-MAP2 antibody (ab5392) was obtained from Abcam (Cambridge CB2 0AX, UK). Anti-rabbit HRP-linked secondary antibody (#7074P2) and goat anti-mouse HRP-linked secondary antibody (#62-6520) were purchased from Sigma-Aldrich and Invitrogen, respectively. Alloxan, CQ, BXZ2, rapamycin, OSMI-1 were dissolved in DMSO. Then, 3-MA was dissolved in DDW.
9
Quantifying Autophagic Flux by LC3 Imaging
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Cells cultured on Lab-Tek II chamber slides were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 2% BSA for 30 min. Cells were then incubated with anti-LC3 antibody (Cell Signaling, Beverley, MA, USA) overnight followed by FITC-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 1 h. DAPI was used for counter-staining. Images were taken with a laser-scanning confocal microscope (Model LSM710, Zeiss, Jena, Germany). LC3 morphology was assessed by an independent viewer in a blind manner. For each independent experiment, 5–10 random high-power fields (at least 50 cells in total) were surveyed. Cells were arbitrarily categorized into punctate LC3+ and LC3- groups (as guidance, LC3 punctation induced by amino-acid starvation was used as a positive control). We also counted the average number of LC3 puncta in individual cells.
10
Autophagy in lung macrophages upon fungal and bacterial stimulation
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For autophagy on lung cells, 1 × 106 purified alveolar macrophages were stimulated on glass slides in 24 multiwell plates with A. fumigatus live conidia or P. aeuginosa and incubated for two at 37 °C in 5% CO2. Cells were incubated with 1:200 diluted anti-LC3 antibody (Cell Signaling Technology) overnight at 4 °C in PBS containing 3% normal bovine serum albumin, incubated with anti-rabbit–PE secondary antibody (Sigma-Aldrich), and fixed for 20 min in PBS containing 4% paraformaldehyde. Images were acquired using the Olympus BX51 fluorescence microscope with a × 40 objective and analySIS image processing software (Olympus). DAPI was used to detect nuclei. An equal amount of cell lysate in 2 × Laemmli buffer (Sigma-Aldrich) was probed with rabbit anti-LC3b-I or anti-LC3b-II antibody (Abcam) for immunoblotting.
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