HEK293 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After incubation with primary antibodies, the cells were subjected to the Duolink in situ PLA according to the manufacturer’s instructions (Duolink In Situ Red, Sigma-Aldrich, duo921001). The following primary antibodies were used: FLAG antibody (Sigma, F3165), PHB2 antibody (Santa Cruz Biotechnology, sc-133094), LC3B antibody (Novus Biologicals, NB1-02200), IgG(M) antibody, and IgG(R) antibody. Images were captured with a Nikon (Ti2) microscope.
Lc3b antibody
Manufactured by Novus Biologicals
Sourced in United States
The LC3B antibody is a primary antibody used for the detection and analysis of the LC3B protein in various biological samples. LC3B is a widely used marker for autophagy, a cellular process that involves the degradation and recycling of damaged or unwanted cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Cisplatin, by Promega (2 mentions)
5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolylcarbocyanine iodide jc 1, by Thermo Fisher Scientific (2 mentions)
Fitc annexin v 7 aad apoptosis detection kit, by BD (2 mentions)
Ldh dependent cytotoxic nonradioactive cytotoxicity assays, by Promega (2 mentions)
N acetylcysteine nac, by Merck Group (2 mentions)
Caspase 3 fluorometric assay kit, by Abcam (2 mentions)
Anti irf 1 antibody, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Flag antibody, by Merck Group (1 mentions)
P62 antibody, by Abcam (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Vector abc kit, by Vector Laboratories (1 mentions)
Rabbit anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Vector dab kit, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Vegfa, by Abcam (1 mentions)
Cd147, by Abcam (1 mentions)
Eclipse e100, by Nikon (1 mentions)
12 protocols using lc3b antibody
1
Duolink PLA for protein interactions
2
Immunoblotting Antibody Identification
3
Immunostaining for Apoptosis and Autophagy
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Sections of 4% paraformaldehyde‐fixed paraffin‐embedded tissue samples, cut at 5‐µm thickness, were used for immunostaining. Staining for cleaved caspase 3 was performed on paraffin sections using a rabbit anti‐cleaved caspase 3 antibody (1:200; Cell Signaling). The secondary antibody was a biotinylated goat anti‐rabbit IgG (1:200; BA‐1000; Vector Laboratories). Staining was performed using the Vector ABC kit and the Vector DAB kit (Vector Laboratories). Staining for LC3BII was performed on paraffin sections using a rabbit polyclonal LC3B antibody (1:200; Novus Biologicals). ImmPRESS HRP Goat Anti‐Rabbit IgG Polymer Kit (Vector Laboratories) was used as a secondary antibody. Staining was performed using a DAB substrate kit (Vector Laboratories).
4
Western Blot Analysis of H9c2 Cells
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H9c2 cells were seeded in 6-well plates and cultured as mentioned above. Cells were lysed in lysis buffer containing protease inhibitors. Protein concentration in the supernatant was determined by the Pierce BCA Protein Assay Kit (Thermo). The samples were subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane, then probed by Western blot analysis with the indicated antibodies, and developed by using an ECL reagent. LC3-b antibody was purchased from Novus Biologicals. Other antibodies were purchased from Sangon Biotech.
5
Western Blot Analysis of Cellular Proteins
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LC3b antibody was purchased from Novus Biologicals. Actin, GAPDH, Bcl2, and PPARα were purchased from Sangon Botech (Shanghai, China). Secondary antibodies were purchased from Jackson Immunoresearch. Western blot method was described previously [7 (link), 17 (link)]. Data are triplicates from three independent experiments.
6
Placental Immunohistochemical Analysis of SARS-CoV-2
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The placenta samples were collected and sent to the Pathology Lab for optimal formalin fixation and histological analysis. One formalin-fixed paraffin-embedded block of placental parenchyma was selected from each case to perform immunohistochemical analyses. After macroscopic examination of the cut surfaces, multiple representative sections of the parenchyma were sampled for histology.
Placenta slides were deparaffined with xylene and rehydrated at decreasing ethanol concentrations. Heat antigen retrieval in Citrate Buffer Ph6 and Ph9, depending on the marker datasheet information, was performed. Slides were stained with the following antibodies, purchased from Abcam: SARS-CoV-2 SPIKE glycoprotein (Cat# ab272504, polyclonal, dilution 1:100), ACE2 (Cat# ab108252, clone EPR4435-2, dilution 1:250), CD147 (Cat# ab666, clone MEM-M6/1, dilution 1:100), CD34 (Cat# ab110643, clone EPR2999, dilution 1:250), and VEGF-A (Cat# ab1316, clone VG-1, 5 µg/ml concentration). LC3B antibody was purchased from Novus Biologicals (Cat# NB100-2220, polyclonal, dilution 1:250). Slides were counterstained with hematoxylin-eosin (H-E) and were imaged with Nikon Eclipse E100 microscope at different magnifications (10 or 20x). The tissues were scored based on number of positively stained cells/mm2 and the staining on selected areas was detected using ImageJ (64-bit Java 8) software.
Placenta slides were deparaffined with xylene and rehydrated at decreasing ethanol concentrations. Heat antigen retrieval in Citrate Buffer Ph6 and Ph9, depending on the marker datasheet information, was performed. Slides were stained with the following antibodies, purchased from Abcam: SARS-CoV-2 SPIKE glycoprotein (Cat# ab272504, polyclonal, dilution 1:100), ACE2 (Cat# ab108252, clone EPR4435-2, dilution 1:250), CD147 (Cat# ab666, clone MEM-M6/1, dilution 1:100), CD34 (Cat# ab110643, clone EPR2999, dilution 1:250), and VEGF-A (Cat# ab1316, clone VG-1, 5 µg/ml concentration). LC3B antibody was purchased from Novus Biologicals (Cat# NB100-2220, polyclonal, dilution 1:250). Slides were counterstained with hematoxylin-eosin (H-E) and were imaged with Nikon Eclipse E100 microscope at different magnifications (10 or 20x). The tissues were scored based on number of positively stained cells/mm2 and the staining on selected areas was detected using ImageJ (64-bit Java 8) software.
7
Western Blot Analysis of Cellular Proteins
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Cells were seeded in 6-well plates and cultured as mentioned above. Cells were lysed in lysis buffer containing protease inhibitors. The lysate was sonicated (10 s) and then centrifuged at 13,000 ×g at 4°C for 10 min. Protein concentration in the supernatant was determined by the Pierce BCA Protein Assay Kit (Thermo). Samples were diluted (1 : 4) in protein loading buffer (67 mM Tris-HCl, pH 6.8, 30% glycerol, 2% SDS, and 0.01% bromophenol blue) and boiled for 5 min. The proteins were separated by SDS-PAGE using 10% acrylamide gels and transferred to a nitrocellulose membrane (PALL). After being blocked with 5% (w/v) nonfat dry milk dissolved in TBST buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% Tween 20), the membranes were incubated with primary antibodies, anti-cascas3/9, anti-PARP-1, Bcl-2, Bax, Bad, and beta-actin (Santa Cruz Biotechnology) and LC3b antibody (Novus Biologicals). The membranes were washed three times with TBST buffer at room temperature and then incubated with HRP-conjugated secondary antibody for 1~2 h at room temperature. The secondary antibody was removed by washing three times with TBST buffer. The immunoreactive bands were visualized with ECL reagent.
8
Western Blot Analysis of LC3B Protein
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For the analysis of LC3B protein, a traditional Western blot was used as the protein was of small molecular weight. Protein concentrations were determined using the Bradford Assay Reagent (Thermo Scientific: cat. no. 23238) and 25 µg of total protein was loaded per well of 4%–20% Mini-Protean TGX gels (Bio-Rad #4561095) and transferred to nitrocellulose membranes (Trans-Blot Turbo Transfer pack, Bio-Rad #1704158) using the Bio-Rad trans-blot turbo transfer system. Antibody against LC3B was used at a dilution of 1:500 (LC3B Antibody; Novus Biologicals: #NB100-2220SS). Secondary antibody was added (1:1,000 Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling #7074s), and the membranes were then developed using an ECL kit. Densitometry analysis was done using ImageJ software and LC3 band density was normalized relative to GAPDH/β-actin (Anti-GAPDH antibody, Rabbit monoclonal, Sigma-Aldrich #SAB5600208) or β-actin (Anti-β Actin antibody, Rabbit monoclonal, Sigma-Aldrich #SAB5600204).
9
Quantifying Autophagy Markers by Western Blot
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Antibodies against LC3-II (NB100–2220, LC3B Antibody Novus Biologicals) and β-Actin (Upstates, Charlottesville, Virginia, USA) were used for detecting the appearance of autophagy. First, the NC membrane was incubated with the antibodies in PBS with 2.5% BSA for 3 h. After washing with PBS for 3 times, the horseradish peroxidase (HRP)-conjugated antibody was added and incubated for another hour. Finally, the antigen-antibody complexes were detected with Immobilion Western HRP Chemiluminescent Substrate (Millipore, USA) and a densitometry apparatus (Appraise, Beckman-Coulter, Brea, California, USA) was used to quantify the blots.
10
Cytotoxicity and Apoptosis Assays
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Cisplatin, methyl thiazolyl tetrazolium (MTT), and LDH-dependent cytotoxic nonradioactive cytotoxicity assays were obtained from Promega (Madison, WI, USA). The LC3B antibody was purchased from Novus Biologicals (Littleton, CO, USA). N-acetylcysteine (NAC), β-actin, and 2,7-dichloro-fluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). An anti-IRF-1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Caspase 3 Fluorometric Assay kit was obtained from BioVision (Milpitas, CA, USA). Goat anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was obtained from Invitrogen (Carlsbad, CA, USA). Assay kits for ATP, superoxide dismutase (SOD), and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Institute. (Nanjing, China). The FITC-Annexin V/7-AAD apoptosis detection kit was obtained from BD Biosciences (USA).
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