Fresh coronal slices containing caudal NTS (300-µm thick) were prepared from 300–500 g male rats (4 rats/group) and incubated with 100 µM FC or Veh for 2 h (the same procedure as for mIPSCs recording). Caudal NTS was dissected from surrounding tissue and each was separately homogenized in the ice-cold Radio-Immunoprecipitation Assay homogenization buffer, sonicated, and centrifuged at 30,000 g for 20 min. Supernatant was collected and protein concentration was measured by bicinchoninic acid assay (Pierce, Cat. No. 23225). Protein (25 µg) from each sample was mixed with 6 µL of SDS-loading buffer dye and boiled for 5 min. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk at room temperature, membranes were incubated with rabbit polyclonal anti-GFAP primary antibody (1:5,000, Abcam, ab7260, Waltham, MA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:10,000, Invitrogen, No. 31460) for 1 h at room temperature. Blots were visualized by enhanced chemiluminescence. Norm-Veh and CIH-Veh were set to 100%. Changes of GFAP/β-actin GeneTex, (GTX629630, Irvine, CA) ratio as a function of FC (100 µM) incubation were compared with their vehicle control and shown as a percentage of control intensity.
Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody is a laboratory reagent used for detection and quantification of target proteins in immunoassays. It contains goat-derived antibodies specific to rabbit immunoglobulin G (IgG) molecules, conjugated with the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Gapdh mab, by Cell Signaling Technology (2 mentions)
Mmp3 pab, by Proteintech (2 mentions)
Anti gfap primary antibody, by Abcam (1 mentions)
Vcx600, by Sonics (1 mentions)
Cox 4, by Abcam (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Glut3, by Abcam (1 mentions)
Glut1, by Proteintech (1 mentions)
Acat1, by Proteintech (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Ab59479, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Luminata crescendo western hrp substrate, by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
11 protocols using horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
1
Western Blot Analysis of GFAP Expression
2
Inducible Probiotic Protein Expression
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Various transformed probiotic clones were cultured in MRS broth, and expression of HLF or BLF was induced under different conditions: LF expression was induced by the addition of 0.1 to 20 ng/mL of nisin for 0 to 8 h. The cell pellets were lysed with an ultrasonic cell disruptor (Sonics & Materials, VCX 600, Newtown, CT, USA) on ice, and cell lysates were analyzed via SDS-PAGE and Western blot analysis. The nitrocellulose membrane for Western blot analysis was incubated with rabbit anti-HLF primary antibody (1:20,000 dilution; Upstate, Cat: 07-685) and subsequently with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Invitrogen, Cat: 65-6120).
3
Quantitative Western Blot Analysis
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Following tissue homogenization and determination of protein concentration, equal amounts of proteins (12 μg/well) were loaded in each well of a 4–20% SDS PAGE gel and transferred onto a 0.2 μm pore size nitrocellulose membrane. Membranes were blocked in 5% blocking solution (Bio-Rad Laboratories, Inc. Hercules, CA) in 0.01% PBS-Tween and immunoblotted overnight (4°C on shaker) with different primary antibodies. Blots were washed with 0.01% PBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ThermoFisher, Waltham, MA) for 1 h at room temperature. After incubation, membranes were washed before visualization using enhanced chemiluminescence (SuperSignal West Femto, ThermoFisher Scientific, Waltham, MA). Primary antibodies used were all rabbit polyclonal: BDH1 (Proteintech, 1:1,000); ACAT1 (Proteintech, 1:1,000); GLUT1 (Proteintech, 1:1,000); GLUT3 (Abcam, 1:2,000); COX IV (Abcam, 1:1,000) and β-actin (Invitrogen, 1:5,000). Band intensities were quantified by densitometry analysis using ImageJ software; corrected for background intensities and normalized to levels of β-actin (soluble fraction) or cytochrome oxygenase IV (COX IV, for mitochondrial enzymes) used as loading controls.
4
Exosomal Protein Characterization via Western Blotting
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Exosomal samples (20 mg) were lysed on ice in 150–250 μL of RIPA buffer (Solarbio, Beijing, China). The samples were then centrifuged (Z 366) at 4°C and 3,000 g for 15 min, and protein concentrations of the supernatants were quantified using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Western blotting was then performed with a Solarbio western blotting kit according to the manufacturer instructions. In brief, the protein samples were electrophoresed on a 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (with 15 μg of protein per well), and the separated proteins were transferred onto polyvinylidene fluoride membranes, which were then blocked with 5% milk in Tris-buffered saline with Tween 20 blocking buffer. The membranes were incubated with antibodies against CD9 (Abcam, ab19715, 1:1000 dilution), CD63 (Abcam, ab59479, 1:1000 dilution), and TSG101 (Abcam, ab125011, 1:1000 dilution) overnight at 4°C. Following incubation with the specific horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, MA1108HRP, 1:1000 dilution), the chemiluminescence signal was detected using ECL western blotting detection reagents (Millipore, WBKLS0100, New York, NY, United States).
5
Quantification of Pancreatic T7 Trypsinogen
6
Western Blot Analysis of Protein Samples
7
Immunoblotting Analysis of P2Y12 and P2Y13 Receptors
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Twenty micrograms of total protein extract from whole brain and spleen tissues were resolved on NuPAGE 4–12% Novex Bis‐Tris Gels (Invitrogen, NP0321) and transferred onto nitrocellulose membranes. Blots were probed with antibodies to P2Y12 (kindly provided by Dr. David Julius, UCSF) and to P2Y13 (Abcam, ab108444) followed by horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:2,000, Thermo Fisher Scientific, 65‐6120). Antibody binding was detected using the Luminata Crescendo Western HRP substrate (Sigma, WBLUR0500). To normalize for protein content, membranes were stripped and re‐probed with anti‐actin antibody (1:5,000, Sigma, A2066). Quantification of the protein expression level was done with ImageJ software using the built‐in Gel Analysis protocol.
8
Western Blot Quantification of MMP3 and GAPDH
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Protein samples were subjected to gel electrophoresis, transferred to a PVDF membrane and blocked with a 5% wt/vol BSA PBST (0.1% Tween-20) solution for 1 h. Membranes were incubated with primary antibodies overnight at 4 °C; MMP3 pAb diluted 1:1,000 (Proteintech Group, 17873-1-AP), GAPDH mAb diluted 1:1000 ,j
(Cell Signaling Technology, 2118S). Membranes were washed with PBST (0.1% Tween-20) and incubated with secondary antibodies for 1 h at room temperature; horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody diluted 1:2,000 (Thermo Fisher Scientific, G-21234). Membranes were washed with PBST (0.1% Tween-20) and imaged with a chemiluminescence reagent (Thermo Fisher Scientific, 34095). Densitometry analyses were performed using ImageJ software.
(Cell Signaling Technology, 2118S). Membranes were washed with PBST (0.1% Tween-20) and incubated with secondary antibodies for 1 h at room temperature; horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody diluted 1:2,000 (Thermo Fisher Scientific, G-21234). Membranes were washed with PBST (0.1% Tween-20) and imaged with a chemiluminescence reagent (Thermo Fisher Scientific, 34095). Densitometry analyses were performed using ImageJ software.
9
Hippocampal Protein Extraction and Quantification
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Hippocampi were homogenized in RIPA lysis buffer (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 5 mM EDTA; 1% NP40; 1% Triton X-100; 1% sodium deoxycholate; 0.1% SDS) with protease inhibitor (1 μL inhibitor: 100 μL RIPA). For protein extraction, HC samples were centrifuged at 13,000 rpm for 17 min under refrigeration (4 °C) and supernatant collected. Protein concentrations were determined by the method of Bradford according to the manufacturer's protocol. SDS polyacrylamide gel electrophoresis (10%) was performed using 50 μg of protein (previously prepared with Laemmli sample buffer and heated at 95 °C for 5 min). The proteins were transferred to PVDF membrane, blocked with BSA 5% for 1 h, and incubated overnight with rabbit anti-parvalbumin IgG primary antibody (1:10,000; Abcam, USA) or mouse anti-α-tubulin IgG primary antibody (1:4000; Sigma, USA). After washing, the blots were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (1:1000; Thermo Scientific, USA) or goat anti-mouse IgG secondary antibody (1:1000; Thermo Scientific, USA) for 90 min at room temperature. Signal was detected using the ECL system (Bio-Rad, USA) according to the manufacturer's instructions, and then the bands were captured with a CCD camera using the ChemiDoc system (Bio-Rad, USA). Densitometry quantification of bands was performed with NIH ImageJ software.
10
Comprehensive Protein Detection Methodology
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Proteins were detected using the following primary antibodies: anti-enterovirus VP1 clone 5-D8/1 antibody purchased from Dako (Denmark); Mouse anti-Ub, Mouse anti-MAT1, Mouse anti-CDK7, and Mouse anti-Cyclin H antibodies purchased from Santa Cruz (USA); Mouse monoclonal to RNA polymerase II CTD and Rabbit monoclonal to RNA polymerase II CTD (phosphor S5) antibodies purchased from Abcam (USA); Mouse anti β-actin purchased from Proteintech; Rabbit monoclonal CDK2 and CDK4, Rabbit monoclonal phosphor-CDK2 and CDK4, Mouse monoclonal pRb, Rabbit monoclonal pRb-phospho Ser780, pRb-phospho Ser795 and pRb-phospho Ser807/811 antibodies purchased from Cell Signaling (USA); Rabbit polyclonal Lamin B1, Mouse monoclonal c-Myc, Mouse monoclonal HA-Tag and Mouse Monoclonal Flag antibodies purchased from Sigma (USA); Goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody purchased from Thermo Fisher Scientific (USA).
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