Cell lysates (30μg) of THP-1 cells treated with AEM-2 and LPS were subjected to SDS-PAGE (15% gel), transferred to nitrocellulose membranes and probed for microtubule-associated protein 1A/1B-light chain 3 (LC3) using a rabbit polyclonal antibody (Sigma Inc). The housekeeping protein β-actin was detected using a rabbit polyclonal antibody (Sigma Inc). After labeling with appropriate secondary antibodies, proteins were visualized using Millipore Immobilon chemiluminescent substrate.
Rabbit polyclonal antibody
Manufactured by Merck Group
Sourced in United States, Germany
Rabbit polyclonal antibody is a laboratory reagent used for the detection and identification of specific target proteins or antigens in various research and diagnostic applications. It is produced by immunizing rabbits with a specific antigen, which stimulates the rabbits' immune system to generate a diverse population of antibodies that recognize different epitopes on the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Horseradish peroxidase conjugated secondary antibody against rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
D mannose agarose, by Merck Group (1 mentions)
Mouse monoclonal antibody against flag, by Merck Group (1 mentions)
Monoclonal anti myc antibody, by Santa Cruz Biotechnology (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
A monoclonal antibody, by Merck Group (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Halotag tmr ligand, by Promega (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Lipofectamine r 2000 reagent, by Thermo Fisher Scientific (1 mentions)
22 protocols using rabbit polyclonal antibody
1
Immunoblotting for Autophagy Marker LC3
2
Protein Analysis by Western Blot
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The western blot analysis of proteins were performed as previously reported [29 (link)]. The antibodies (NOX2, JNK, ERK, and p38) were purchased from Beijing Bioss Biotechnology Co. Ltd. The dilution ratio of primary antibodies is shown in Table S2 . The GAPDH content was analyzed as the loading control with rabbit polyclonal antibody (Sigma-Aldrich, USA).
3
Western Blot Analysis of Apoptosis Markers
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The protein samples were separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 12 h and incubated for 1 h at 37°C with the following diluted primary antibodies: TNFRSF1B (1/100), c-Jun N-terminal kinase (JNK) (1/100), caspase3 (1/500), Bax (1/500) and Bcl-2 (1/100). After washing four times for 5 min each with PBST, the membranes were incubated for 1 h at 37°C with peroxidase-conjugated secondary antibodies against rabbit IgG (1/2000, Santa Cruz, USA). After washing four times for 5 min each, the bound antibodies were visualized by chemiluminescence using the ECL-plus reagent (GE Healthcare, Buckinghamshire, UK). The β-actin content was analyzed as the loading control with rabbit polyclonal antibody (Sigma, USA).
4
Antibody Validation Protocol
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Rabbit polyclonal antibody and mouse monoclonal antibody against FLAG were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-Myc antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-AAT antibody was purchased from Proteintech (Rosemont, IL). Rabbit monoclonal anti-LMAN1 antibody was purchased from Abcam (Cambridge, MA). Protein A/G PLUS-Agarose beads were purchased from Santa Cruz Biotechnology. D-mannose agarose was purchased from Sigma-Aldrich. Human AAT enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (Minneapolis, MN).
5
Western Blot Analysis of TREK-1 Protein
6
Immunohistochemical Analysis of GGH
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Freshly-cut TMA sections were immunostained on one day and in one experiment. Slides were deparaffinised and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 7.8 Tris-EDTA-citrate buffer. A primary antibody specific for GGH (rabbit polyclonal antibody, Sigma-Aldrich, Munich, Germany; cat#HPA025226; dilution 1:450) was applied at 37 °C for 60 min. The bound antibody was then visualized using the EnVision Kit (Dako, Glostrup, Denmark) according to the manufacturer’s directions. GGH typically stained the cytoplasm in all (100%) tumor cells of a tissue spot. Accordingly, the staining intensity was recorded in three categories for each cancer, including negative (no detectable staining), low (weak to moderate staining), and high (strong staining) by independent evaluation by at least two scorers.
7
Western Blot Analysis of TREK-1 Protein
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The uterine strips were homogenized in RIPA lysis buffer (Beyotime Biotechnology) and centrifuged at 10000 g for 5 min. After centrifugation, the protein concentration in the supernatant was measured using BCA protein assay kit (Beyotime Biotechnology). Samples were subjected to electrophoresis on 10% SDS polyacrylamide gel, then protein was transferred electrophoretically from gels to PVDF membranes (Millipore). The membranes were blocked with 5% nonfat dry milk in PBS-Tween buffer for 1 h, and then incubated overnight at 4°C with antibody solution containing TREK-1 (1:1000) rabbit polyclonal antibody (Sigma). GAPDH was used as an internal control and detected by a monoclonal antibody (1:500000, Sigma). After washing in PBS-Tween, the PVDF membranes were incubated in horseradish peroxidase conjugated secondary antibody (1:5000) for 1.5 h. The membrane blots were washed with PBS-Tween and visualized with enhanced chemiluminescence (ECL). The reactive band corresponding to TREK-1 was analyzed by optical densitometry and ImageJ software (National Institutes of Health). The densitometry value represented the pixel intensity normalized to GAPDH to correct for loading.
8
Immunocytochemical Labeling of tPA Expressing Neurons
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Recombinant human tPA (Actilyse®) was purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany). Fetal bovine serum, horse serum, lipofectamine R 2000 reagent, B27 supplement, glutamine, laminin, neurobasal medium, and penicillin/streptomycin were purchased from ThermoFisher (Waltham, Massachusetts, USA). Dulbecco’s modified Eagle’s medium (DMEM), poly-D-lysine, phosphate-buffered saline (PBS), Glycine (Gly), paraformaldehyde, albumin from bovine serum, ammonium chloride (NH4Cl), potassium chloride (KCl), and rabbit polyclonal antibody were purchased from Sigma-Aldrich (St Louis, MO, USA). Tetrodotoxin citrate (TTX), cyanquixaline (CNQX), (2R)-amino-5-phosphonovaleric acid (APV) and bicuculline methiodide (Bic) were purchased from Tocris (Bristol, UK). HaloTag® TMR ligand was purchased from Promega (Madison, Wisconsin, USA). The following primary antibodies were used for immunocytochemistry: mouse monoclonal anti-HaloTag® (dilution 1:1,000; Promega; G9211) rabbit anti-tPA polyclonal antibody (dilution 1:1 500; generous gift from R. Lijnen, Leuven), chicken anti-Microtubule-associated protein 2 (MAP2) polyclonal antibody (dilution 1:8 000; Abcam, Cambridge, UK; ab5392). Secondary fluorescent antibodies (Alexa647; dilution 1:800) were purchased from Jackson Immunoresearch (Bar Harbor, ME, USA).
9
Tracking HUVEC Migration and Collagen-I Expression
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To investigate the migration of Human Umbilical Vein Endothelial Cells (HUVEC, Beijing Yuhengfeng Biotech Co. Ltd.) on samples, we used a microfluidic chip. The HUVEC was stained by Cell Tracker-green (Thermofisher Scientific, USA). The microfluidic chips were fixed on the surface of samples. Then 3 × 106 HUVEC suspension was injected into the microfluidic channel, then put into the incubator culturing for 4 h and peeled off microchip. The patterned cells on samples were observed by laser confocal microscopy at 0, 3, 6, 12 and 24 h. The reduction of the blank area is seen as the wound healing process.
To investigate the expression of Collagen-I, HUVEC were seeded on different samples and cultured 7 days. Then HUVEC were fixed by 3.7% formaldehyde, permeabilized with a rupture agent for 5 min, incubated with closed fluid for 1 h, primary antibody (Sigma-Aldrich, American) for 2 h at 37 °C. After that, the cells were incubated with rabbit polyclonal antibody (Sigma-Aldrich, American) for another 1 h. The nuclei were stained with Hochest 33342 (Dojindo, Japan), and observed with laser confocal microscopy.
To investigate the expression of Collagen-I, HUVEC were seeded on different samples and cultured 7 days. Then HUVEC were fixed by 3.7% formaldehyde, permeabilized with a rupture agent for 5 min, incubated with closed fluid for 1 h, primary antibody (Sigma-Aldrich, American) for 2 h at 37 °C. After that, the cells were incubated with rabbit polyclonal antibody (Sigma-Aldrich, American) for another 1 h. The nuclei were stained with Hochest 33342 (Dojindo, Japan), and observed with laser confocal microscopy.
10
Western Blot Analysis of Apoptosis Signaling
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Total protein was assessed by SDS-polyacrylamide gel electrophoresis under reducing conditions on 12% gels and then was transferred to nitrocellulose membranes using a tank transfer at 80 mV in Tris-glycine buffer containing 20% methanol for 12 h at 4 °C. Nitrocellulose membranes were blocked for 1 h with 5% bovine serum albumin (BSA) at 37 °C and were incubated for 12 h with the following antibodies: RNF11 (1:800), RIPK1 (1:1500), RIPK3 (1:1500), Caspase8 (1:1000), MLKL (1:1000), JNK (1:2000), ERK (1:1000), p38 (1:1000), PhosphoPlus JNK (1:1000), PhosphoPlus ERK (1:2000) and PhosphoPlus p38 (1:1000) at 4 °C. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:1500, Santa Cruz, CA, USA) using the ECL kit (Kangweishiji Biotechnology, Beijing, China). The GAPDH content was analyzed as the loading control with rabbit polyclonal antibody (Sigma, USA).
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