PE was purchased from Tokyo Chemical Industry (P0398). The autophagy inhibitor CQ was obtained from Sigma‐Aldrich, St Louis, MO, USA (C6628) and was applied to cardiomyocytes at a concentration of 10 μM 16 . Protein G‐Agarose was purchased from Roche (Roche Diagnostics, Mannheim, Germany). For the Western blotting detection of specific proteins, the following primary antibodies obtained from Cell Signaling Technology (Danvers, MA, USA) were used: anti‐Atg7 (#2631), anti‐AMPK (Thr172) (#2531), anti‐AMPK (#2532), antiphosphorylated mTOR (Ser2448) (#5536), antiphosphorylated p70S6K (Thr389) (#9234), anti‐p70S6K (#2708), antiphosphorylated Akt (Ser473) (#4058), anti‐Akt (#9272) and anti‐GAPDH (#2118) antibodies. Anti‐Sestrin 1 antibody was obtained from Abcam, Cambridge, UK (ab134091). Anti‐LC3B antibody was obtained from Novus Biologicals, Littleton, CO, USA (NB100‐2220). Anti‐p62 antibody was purchased from Sigma‐Aldrich (P0068). Antivinculin antibody was purchased from Sigma‐Aldrich (V9264). Anti‐cathepsin D antibody was obtained from Santa Cruz Biotechnology, Dallas, TX, USA (sc‐6486).
Anti lc3b antibody
Manufactured by Novus Biologicals
Sourced in United States
The Anti-LC3B antibody is a laboratory tool used to detect and study the LC3B (Microtubule-associated protein 1A/1B-light chain 3) protein. LC3B is a well-known marker for autophagy, a cellular process involved in the degradation and recycling of cellular components. This antibody can be used in various immunological techniques, such as Western blotting, immunohistochemistry, and immunofluorescence, to visualize and quantify the expression of LC3B in different samples and experimental conditions.
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Lab products found in correlation
Diamidine phenylindole dapi, by Thermo Fisher Scientific (3 mentions)
Dmi6000b, by Leica (3 mentions)
Triton x 100, by Keygen Biotech (3 mentions)
Jem 1400 electron microscope, by JEOL (2 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti atg7, by Cell Signaling Technology (1 mentions)
Anti ampk thr172, by Cell Signaling Technology (1 mentions)
Anti phosphorylated mtorser2448, by Cell Signaling Technology (1 mentions)
Antiphosphorylated p70s6k thr389, by Cell Signaling Technology (1 mentions)
Antiphosphorylated akt ser473, by Cell Signaling Technology (1 mentions)
Anti p62 antibody, by Merck Group (1 mentions)
Anti cathepsin d antibody, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti vinculin antibody, by Merck Group (1 mentions)
Anti ampk, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protein g agarose, by Roche (1 mentions)
Anti e2f1 antibody, by Santa Cruz Biotechnology (1 mentions)
Carl lsm 780 microscope, by Zeiss (1 mentions)
Anti p62 antibody, by Abcam (1 mentions)
10 protocols using anti lc3b antibody
1
Autophagy Modulation in Cardiomyocytes
2
Cytosolic Area and Autophagy Analysis
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The experiments are performed as our previous described procedures.39 (link) EM images were obtained from thin sections using a JEM1400 electron microscope (JEOL, Akishima, Tokyo, Japan). The relative cytosolic areas in cross-sections of 10 cells were analyzed by Image-Pro Plus software. And fluorescence signals obtained using anti-LC3B antibody (Novus Biologicals), anti-E2F1 antibody (Santa Cruz), anti-ULK1 antibody (Novus Biologicals) and anti-DRAM2 antibody (Sigma-Aldrich) were acquired and analyzed by Carl Zeiss LSM 780 microscope (Carl Zeiss, Jena, Germany). At least 10 cells from each group were analyzed.
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from the cell lysates using RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, 1:50 dilution). Protein concentrations of the extracts were measured with a BCA assay kit. For Western blot, samples were mixed with sodium dodecyl sulfate (SDS) loading buffer and separated on 8%,10%, 12%, or 15% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were probed overnight at 4 °C with the following primary antibodies: anti-α-synuclein antibody (Abcam, Cambridge, UK, ab138501, 1:1000 dilution); anti- p62 antibody (Abcam, ab91526, 1:1000 dilution); anti-LC3B antibody (Novus, St. Charles, MO, USA, NB100-2220, 1:1000 dilution); anti-HDAC4 antibody (Proteintech, Wuhan, China, 17449-1-AP, 1:600 dilution); anti-β-actin antibody (Antgene, Wuhan, China, ANT009, 1:1000 dilution). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution) for 1 h at room temperature, western blots were revealed through chemiluminescence. The protein levels were quantified through densitometry using ImageJ v1.47 software.
4
Autophagy and Apoptosis Pathway Analysis
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mASMCs were rinsed with ice-cold PBS once and then lysed in RIPA lysis buffer. The protein concentration of the sample was determined by Bio-rad ABS kit. 20 µg of the protein samples were loaded to each lane and separated in 12% SDS-PAGE gel, then blotted to PVDF membrane. The membrane was incubated overnight with anti-LC3B antibody (Novus; 1:1000 dilution) or anti-p62/SQSTM1 (1:1000; abcam, ab91526) for autophagy detection, or with anti-TRPM2 antibody (Abcam, ab11168, 1:1000 dilution) for expression study, or with anti-caspase-3 antibody (Cell Signaling Technology; 1:1000 dilution) for cell apoptosis detection, or with anti-ATP6V0A1 (Proteintech, 1:1000) for detection of vacuolar H+-ATPase. Immunodetection was accomplished with horseradish-conjugated secondary antibodies, followed by ECL detection system (Amersham Pharmacia).
5
Autophagic Marker Detection in MSCs
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We detected a specific autophagy marker for LC3-positive MSCs using immunofluorescence staining. MSCs were washed twice with PBS. Then, they were fixed with 4% paraformaldehyde in PBS for 30 min, and they were permeabilized with methanol. The MSCs were treated with 3% hydrogen peroxide in methanol for 15 min and washed with tap water for 15 min. The cells were incubated with 0.5% Triton X-100 (KeyGEN) for 5 min, and then, they were washed and incubated with blocking solution (10% goat serum in PBS) for 30 min. Next, they were treated with anti-LC3B antibody (1:200 dilution; Novus Biological, Littleton, CO, USA) overnight at 4 °C. After washing with blocking solution 3 times, the cells were incubated with secondary antibodies. The cells were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA), and they were imaged using a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany).
6
Visualizing Autophagy in Mesenchymal Stem Cells
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Transmission electron microscopy (TEM) (JEM-1011, JEOL, Akishima, Tokyo, Japan) was used to observe the autophagosomes of MSCs, as described previously.14 (link)
The morphological characteristics of autophagosomes include a crescent or cup-shaped bilayer or multilayer membrane, with a tendency to enclose cytoplasmic components. Immunofluorescence analysis was performed to detect a specific autophagy marker LC3, as described previously.14 (link)
Briefly, the cells were washed with phosphate-buffered saline (PBS) (KeyGEN) and fixed with 4% paraformaldehyde. Then, the cells were again washed with PBS and incubated with 0.5% Triton X-100 (KeyGEN). Subsequently, the cells were incubated with a blocking solution (10% goat serum in PBS) and then treated with the anti-LC3B antibody (Novus Biological, Littleton, CO, USA) overnight at 4 °C. Finally, the cells were incubated with secondary antibodies after washing with the blocking solution. The MSCs were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA) and observed under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany). Western blot analysis was used to detect the autophagy makers Beclin1 and LC3, as described previously.14 (link)
The morphological characteristics of autophagosomes include a crescent or cup-shaped bilayer or multilayer membrane, with a tendency to enclose cytoplasmic components. Immunofluorescence analysis was performed to detect a specific autophagy marker LC3, as described previously.14 (link)
Briefly, the cells were washed with phosphate-buffered saline (PBS) (KeyGEN) and fixed with 4% paraformaldehyde. Then, the cells were again washed with PBS and incubated with 0.5% Triton X-100 (KeyGEN). Subsequently, the cells were incubated with a blocking solution (10% goat serum in PBS) and then treated with the anti-LC3B antibody (Novus Biological, Littleton, CO, USA) overnight at 4 °C. Finally, the cells were incubated with secondary antibodies after washing with the blocking solution. The MSCs were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA) and observed under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany). Western blot analysis was used to detect the autophagy makers Beclin1 and LC3, as described previously.14 (link)
7
Autophagosome Visualization by TEM and IF
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TEM images of autophagosomes were obtained from thin sections using a JEM1400 electron microscope (JEOL, Japan). The autophagy fluorescence signals were obtained using an anti-LC3B antibody (Novus Biologicals, Taiwan of China) analyzed by Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).
8
Immunoblotting and Antioxidant Assays
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Anti-LC3B antibody was purchased from NOVUS Biologicals (Littleton, CO, USA); anti-p62 antibody from Abnova (Taipei, Taiwan); anti-GRP78 antibody, from Abcam (Cambridge, MA, USA); anti-CHOP, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, phospho-mTOR, caspase-3, phospho-Akt antibodies from Cell Signaling Technology (Danvers, MA, USA) Bax and PARP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies from Invitrogen (Carlsbad, CA, USA). Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), tert-butyl hydroperoxide (t-BHP), epigallocatechin-gallate (EGCG), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ethanol (EtOH), methanol (MeOH), sodium carbonate, aluminum chloride (AlCl3), Folin–Ciocalteu (F-C)’s phenol reagent, acetic acid, gallic acid, epicatechin, rutin, tannic acid, naringin, quercetin, and β-actin antibody were acquired from Sigma-Aldrich (St. Louis, MO, USA).
9
Antibody Panel for Protein Analysis
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The following primary antibodies were used: anti-FLAG antibody (Sigma), anti-GFP antibody (Santa Cruz), anti-GAPDH antibody (Millipore), anti-phospho-p70S6K (T389) antibody (Cell Signaling Technology), anti-p70S6K antibody (Epitomics), anti-mTOR antibody (Cell Signaling Technology), anti-LC3B antibody (Novus Biologicals), anti-LC3B antibody (Abcam), anti-GAPDH antibody (Proteintech), anti-Tubulin antibody (Proteintech), anti-MAP2 antibody (Proteintech), anti-LAMP2 antibody (Santa Cruz), anti-Bcl2 (total, pS70 or pS87) antibody (Santa Cruz) and anti-BECN1 antibody (Santa Cruz). The following secondary antibodies were used: horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) for immunoblotting and Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Mouse IgG antibody (Proteintech), Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG antibody (Proteintech) or Alexa Fluor 647-conjugated AffiniPure Donkey Anti-Rabbit IgG antibody (Yasen Biotechnology) for immunocytochemistry and immunohistochemistry. DAPI (Sigma) or Hoechst (Sigma) were used for nuclei staining.
10
Quantifying Autophagy in MSCs via LC3B Immunostaining
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The MSCs were seeded onto slides and cultured for 10 days. And then the MSCs were fixed with 4% paraformaldehyde for 30 min on ice, washed twice with PBS, and incubated with 3% H2O2-methanol solution at room temperature for 10 min. Micromass pellets were washed twice with PBS, fixed for 24 h in 10% formalin, embedded in paraffin, and cut into 5-μm thick sections. The latter were deparaffinized, rehydrated, and then washed with PBS. After a 5-min incubation with 0.5% Triton X-100 (KeyGEN), the cells/sections were blocked with 10% goat serum in PBS for 30 min and incubated overnight with anti-LC3B antibody (1:200; Novus Biological) at 4 °C. The slides were washed thrice with the blocking solution, incubated with fluorochrome-conjugated secondary antibody, and counterstained with diamidine phenylindole (DAPI; Molecular Probes, Waltham, MA, USA). The LC3 punctae were observed and counted under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany) [25 (link), 26 (link)].
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