Immunohistochemistry was conducted in 10% formalin-fixed, paraffin-embedded tissues. Dissected tissues were cleansed twice with distilled water, and residual fixatives were removed by 1 hour treatment with 1% sodium. The tissues were pre-treated with 3% hydrogen peroxide for 10 minutes, cleansed with distilled water; and cultured for 5 minutes with 1 TBST (Tris-buffered 5 saline with 0.1% Tween 20). To prevent nonspecific reactions, tissues were treated with normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 hour, and then cultured overnight with rabbit anti-AHR (1:300; Abcam, Cambridge, MA, USA), rabbit anti-CYP1A1 (1:300; Abcam), and rabbit anti-LC3 (1:300; Novus Biologicals, Littleton, CO, USA). Tissues were cleansed with 1 TBST, and cultured with biotinylated secondary antibody solution from the Dako REAL EnVision Detection System (Dako, Glostrup, Denmark) for 30 minutes at room temperature. The tissues then then cleansed with distilled water; counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA); dehydrated and clarified based on a conventional method; and then prepared for Leica microsystem DFi8 LASX software light microscopy (Leica, Wetzlar, Germany).
Rabbit anti lc3
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Rabbit anti-LC3 is a primary antibody that recognizes the microtubule-associated protein 1 light chain 3 (LC3) protein. LC3 is a key marker for autophagy, a cellular process involved in the degradation and recycling of cellular components. This antibody can be used to detect and quantify LC3 protein levels in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti mtor, by Cell Signaling Technology (3 mentions)
Bafilomycin a1, by Merck Group (3 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Mouse anti lamp1, by Santa Cruz Biotechnology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Rabbit anti β actin, by Cell Signaling Technology (2 mentions)
Rabbit anti beclin 1, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Dako real envision detection system, by Agilent Technologies (1 mentions)
Rabbit anti ahr, by Abcam (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Mito tempo, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti tom20, by Santa Cruz Biotechnology (1 mentions)
21 protocols using rabbit anti lc3
1
Immunohistochemical Analysis of AHR, CYP1A1, and LC3
2
Antiviral Autophagy Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells, HeLa cells, U2OS cells and Vero cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT, USA) and antibiotics (100 U ml−1 penicillin and 100 μg ml−1 streptomycin, Invitrogen). Antibodies used in this study for western blot or immunofluorescence assays were as follows: mouse anti-Flag (Sigma, St Louis, MO, USA), rabbit anti-LC3 (Novus, Littleton, CO, USA and MBL, Woburn, MA, USA), mouse anti-TIM23 (BD, Waltham, MA, USA), mouse anti-GAPDH and rabbit anti-Caspase-3 (Sungene Biotech, Tianjin, China), rabbit anti-Myc and rabbit anti-HA (MBL), rabbit anti-ATG5 (Epitomics, Cambridge, MA, USA), and rabbit anti-TOM20, mouse anti-Calnexin, mouse anti-TRAF2 and rabbit anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Cleaved Caspase-3 (Cell Signalling Technology, Danvers, MA, USA); the antibody against MAVS was produced by our lab [14 (link)]. Mito-TEMPO was purchased from Santa Cruz Biotechnology or Enzo Life Science, Farmingdale, NY, USA. Pyrrolidinedithiocarbamic acid ammonium salt (PDTC), bafilomycin A1 and chloroquine were purchased from Sigma. VSV was propagated in Vero cells.
3
Molecular Mechanisms of Inflammatory Signaling
4
Statin-Induced Autophagy and Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Atorvastatin, mevalonate, bafilomycin A1 and rapamycin (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at an appropriate stock concentration. The following primary antibodies were used in this study: rabbit anti-procaspase-3 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PARP (1:1000, Cell Signaling Technology), rabbit anti-LC3 (1:2000 for western blot and 1:400 for immunocytochemistry, Novus biological, Littleton, CO, USA), mouse anti-p62 (1:2000, Becton, Dickinson and Company (BD) biosciences, San Jose, CA, USA) and Horseradish Peroxidase (HRP)-conjugated actin (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA).
5
Antibody selection and usage in cellular analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies (at the dilution indicated) were used for either immunofluorescence or Western blotting: rabbit anti-Ki67 (1:100, cod. HPA001164; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-p21 (1:100, cod. B1313; Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-Histone H3 (1:500, cod. 61475; Active motif, Carlsbad, San Diego, CA, USA); mouse anti-LC3 (1:100, cod. NB600-1384; Novus Biologicals, Milano, Italy); rabbit anti-LC3 (1:1000, cod. L7543; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-LAMP1 (1:1000, cod. 555798; BD, Biosciences, Franklin Lakes, NJ, USA); rabbit anti-β-Catenin (1:500, cod. PA5-77934; Invitrogen, Paisley, UK); rabbit-anti ATG7 (1:500, cod. AB10511; Millipore, Burlington, MA, USA); mouse-anti ATG7 (1:500, cod. SAB4200304; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β-tubulin (1:1000, cod. T5326; Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-GAPDH (1:1000, cod. G9545; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich, St. Louis, MO, USA).
6
Antibody Validation for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were purchased commercially: mouse anti-Mdm2 2A10 (University of North Carolina Tissue Culture and Molecular Biology Support Facility), mouse anti-actin (Neomarkers), mouse anti-p53 DO.1 (Neomarkers), rabbit anti-Rap2B (Abcam), and goat anti-p21 (C-19) (Santa Cruz Biotechnology), rabbit anti-LC3 (Novus Biologicals).
7
Quantifying Cytoskeleton and Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents used were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands) unless specified otherwise. The primary antibodies and probes used for monolayer assessment were Phalloidin-AF488 (Invitrogen, Carlsbad, CA, USA) diluted 1:100, Mouse anti-α-tubulin (#EP1332Y; Invitrogen, Carlsbad, CA, USA) diluted 1:500 and rabbit anti-Na+/K+ATP-ase (a kind gift from Dr. Jan Koenderink, Radboudumc, The Netherlands) diluted 1:500. The primary antibodies used for autophagy and vesicle trafficking assessment were rabbit anti-LC3 (Novus Biologicals, Abingdon, UK #NB600-1384SS) diluted 1:1000, mouse anti-P62 (SQSTM1) (#610832; BD Biosciences, Mississauga, ON, Canada) diluted 1:1000, mouse anti-LAMP1 (#sc-18821; Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200, and rabbit anti-mTOR (#2983; Cell Signaling Technology, Leiden, The Netherlands) diluted 1:400. The secondary antibodies used for detection were Polyclonal goat anti-rabbit (#P0448, Dako products, Carpinteria, CA, USA) diluted 1:5000, and polyclonal goat anti-mouse (#P0447, Dako products, Carpinteria, CA, USA) diluted 1:5000. Additionally, Alexa-488 goat anti- mouse (#ab150113; diluted 1:500), Alexa-647 goat anti-rabbit (#ab150083; diluted 1:200), donkey anti-rabbit (#AF647; diluted 1:300), and donkey anti-mouse (#AF568; diluted 1:200) secondary antibodies were all from Abcam (Amsterdam, The Netherlands).
8
Autophagic Flux Analysis in WT and KO Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS WT, ATG7KO, HeLa WT, and HeLa p62KO were seeded into a 12-well plate for 24 h. Cells were then washed twice with PBS and treated for 2 h with EBSS (ThermoFischer, #24010043) and/or 200 nM Bafilomycin A1. For LC3 immunoblotting with rapalog2 treatment, HeLa cells were treated for 4 h with 500 nM rapalog2. Finally, cells were lysed and processed for WB.
Primary antibodies used were: rabbit anti-LC3 (Novus Biologicals, #NB600-1384, 1/1000), mouse anti-p62 (Abcam, #ab56416, 1/1000),mouse anti-actin (Merck, #MAB1501, 1/5000), and mouse anti-tubulin alpha (Sigma, #T5168, 1/20,000). Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 680/800 were used for visualization and purchased from ThermoFischer Scientific.
Primary antibodies used were: rabbit anti-LC3 (Novus Biologicals, #NB600-1384, 1/1000), mouse anti-p62 (Abcam, #ab56416, 1/1000),mouse anti-actin (Merck, #MAB1501, 1/5000), and mouse anti-tubulin alpha (Sigma, #T5168, 1/20,000). Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 680/800 were used for visualization and purchased from ThermoFischer Scientific.
9
Mitochondrial Dynamics and Oxidative Stress Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitotracker® Red CMXRos (Thermo Fisher Scientific) to visualize mitochondrial networks in live mGECs [34 (link)].
mGECs on coverslips were fixed with methanol and incubated with anti-8-oxoG monoclonal antibody (N45.1; Japan Institute for the Control of Aging) previously described [35 (link)]. Cells were also stained with either goat anti-mtTFA antibodies (Santa Cruz), rabbit anti-LC3 (Novus Biologicals), or rabbit anti-phospho-histone H2A.X (Ser139) Antibody (Cell Signaling Technology). The antigen-antibody complexes were visualized with Alexa Fluor secondary antibodies. DAPI was added to mounting medium. mGECs plated in 12-well slide (Ibidi) at a density of 2.0 × 104 cells/well and were transfected with WT and mutant GFP-endoG and fixed with 4% PFA in PBS. DAPI was added to mounting medium. Podocytes on coverslips were fixed with 4% PFA in PBS, incubated anti-Caspase 3 antibodies (cleaved Santa Cruz) and Phalloidin-FITC (Sigma). DAPI was added to mounting medium. The cells were imaged with a Zeiss Axioplan2 equipped with Q-imaging MP3.3 RTV color camera running QED capture software.
mGECs on coverslips were fixed with methanol and incubated with anti-8-oxoG monoclonal antibody (N45.1; Japan Institute for the Control of Aging) previously described [35 (link)]. Cells were also stained with either goat anti-mtTFA antibodies (Santa Cruz), rabbit anti-LC3 (Novus Biologicals), or rabbit anti-phospho-histone H2A.X (Ser139) Antibody (Cell Signaling Technology). The antigen-antibody complexes were visualized with Alexa Fluor secondary antibodies. DAPI was added to mounting medium. mGECs plated in 12-well slide (Ibidi) at a density of 2.0 × 104 cells/well and were transfected with WT and mutant GFP-endoG and fixed with 4% PFA in PBS. DAPI was added to mounting medium. Podocytes on coverslips were fixed with 4% PFA in PBS, incubated anti-Caspase 3 antibodies (cleaved Santa Cruz) and Phalloidin-FITC (Sigma). DAPI was added to mounting medium. The cells were imaged with a Zeiss Axioplan2 equipped with Q-imaging MP3.3 RTV color camera running QED capture software.
10
Quantifying Autophagy Biomarker LC3II/LC3I
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of autophagy biomarker LC3II/LC3I, 3 × 105 cells (MDA, HUVEC and J774) were seeded into a 6 well plate. After 24 h, each cell line was treated with the same amount (1 ng/cell) of MSN and Br–MSN for 24 h. Nontreated cells were used as controls. Cells were lysed and proteins were extracted using RIPA buffer (Thermo) with protease inhibitor cocktail (Thermo) and PMSF at 0.5 mM.
Twenty micrograms of whole cell lysate was loaded onto a 12% SDS polyacrylamide gel and then transferred on a PVDF membrane for Western blotting analysis. The membranes were blocked in 5% nonfat milk in TTBS for 2 h and then incubated overnight at 4 °C with rabbit anti-LC3 (Novus Biologicals; 1:2000) and mouse anti-GAPDH (Santa Cruz Technologies; 1:200) primary antibodies. Then membranes were incubated with HRP (horseradish peroxidase)-conjugated anti-rabbit and anti-mouse IgG (Sigma–Aldrich) secondary antibodies, respectively. The bands were detected by chemiluminescence using the SuperSignal West Dura Chemiluminescent Substrate (Thermo); images were visualized, acquired and analyzed with ChemiDoc XRS+ System and Image Lab software (Bio-Rad).
Twenty micrograms of whole cell lysate was loaded onto a 12% SDS polyacrylamide gel and then transferred on a PVDF membrane for Western blotting analysis. The membranes were blocked in 5% nonfat milk in TTBS for 2 h and then incubated overnight at 4 °C with rabbit anti-LC3 (Novus Biologicals; 1:2000) and mouse anti-GAPDH (Santa Cruz Technologies; 1:200) primary antibodies. Then membranes were incubated with HRP (horseradish peroxidase)-conjugated anti-rabbit and anti-mouse IgG (Sigma–Aldrich) secondary antibodies, respectively. The bands were detected by chemiluminescence using the SuperSignal West Dura Chemiluminescent Substrate (Thermo); images were visualized, acquired and analyzed with ChemiDoc XRS+ System and Image Lab software (Bio-Rad).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!