SH-SY5Y cells were used to test the effect of metals on mammalian TH phosphorylation at Ser40. Antibodies of anti-tyrosine hydroxylase (AB152) and anti-tyrosine hydroxylase, phosphoSer 40 (AB5935) were purchased from Sigma-Aldrich (Shanghai, China). The secondary antibody HRP-conjugated goat anti-rabbit IgG was purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). For Western blot analysis, cells were homogenized in the buffer containing 1% Triton X-100 plus 10% proteinase inhibitor cocktail (Sigma), centrifuged, separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Watford, UK). Signals were developed with the ECL detection kit (Vigorous Biotechnology, Beijing, China). The experiments were repeated three times.
Hrp conjugated goat anti rabbit igg
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
HRP-conjugated goat anti-rabbit IgG is a secondary antibody that is conjugated to horseradish peroxidase (HRP). This antibody is used to detect and quantify rabbit immunoglobulin G (IgG) in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab152, by Merck Group (1 mentions)
Ecl detection kit, by Vigorous Biotechnology (1 mentions)
3 3 diaminobezidine, by Agilent Technologies (1 mentions)
Tnf α, by Abcam (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Phospho stat1 tyr701, by Cell Signaling Technology (1 mentions)
Phospho jak2, by Cell Signaling Technology (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Ab63097, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Anti lc3a b, by Cell Signaling Technology (1 mentions)
Dab detection kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
7 protocols using hrp conjugated goat anti rabbit igg
1
Mammalian Tyrosine Hydroxylase Phosphorylation
2
Immunohistochemical Evaluation of NAT10 Expression
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Sections (4 μm thick) were dewaxed in xylene and gradually rehydrated gradually. After antigen retrieval, endogenous peroxidases were blocked with 3% hydrogen peroxide. Then, the sections were incubated with 10% goat serum for 30 min at room temperature. Sections were incubated with rabbit anti-NAT10 polyclonal antibody at 4 °C overnight and then with HRP-conjugated goat anti-rabbit IgG (Zhongshan Golden bridge Biotechnology, Beijing, China) at 37 °C for 30 min. Immunocomplexes were visualized by using 3,3-diaminobezidine (DAKO, CA, USA). Slides were counterstained with light hematoxylin, dehydrated, and cover-slipped.
Histological slides were assessed by two independent observers, including an experienced pathologist, blinded to all clinical, pathological, and outcome information. The score discrepancies were discussed to achieve a consensus. Immunostaining was categorized into four groups: negative (0 score), 0%–10% positive cells; faintly positive (1 score), 10%–25% positive cells; moderately positive (2 scores), 25–50% positive cells; and highly positive (3 scores), ≥50% positive cells.
Histological slides were assessed by two independent observers, including an experienced pathologist, blinded to all clinical, pathological, and outcome information. The score discrepancies were discussed to achieve a consensus. Immunostaining was categorized into four groups: negative (0 score), 0%–10% positive cells; faintly positive (1 score), 10%–25% positive cells; moderately positive (2 scores), 25–50% positive cells; and highly positive (3 scores), ≥50% positive cells.
3
Corneal Tissue Protein Analysis
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The corneal tissues were lysed with cold RIPA buffer, and the total protein concentration was subjected to electrophoresis on an 10% SDS-PAGE gel with an equal amount. After being transferred to polyvinylidene fluoride (PVDF) membranes and blocked by 5% evaporated milk, proteins were incubated overnight with primary antibodies at 4°C. The following primary antibodies were used: phosphorylated nuclear factor-κB p65 (pNF-κB p65, 1 : 1000 Cell Signaling Technology, Danvers, MA, USA), NF-κB p65 (1 : 1000, Cell Signaling Technology), TNF-α (1 : 1000, Abcam, Cambridge, UK), and β-actin (1 : 10000; Sigma, St. Louis, MO) was used as a loading control. HRP-conjugated goat antirabbit IgG (1 : 3000; Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) was used as the secondary antibody. Densitometry of the Western blot bands was performed using the Image J software.
4
Western Blot Analysis of Signaling Pathways
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Total cell lysates were prepared and protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Beyotime, Beijing, China). Cell extracts were subjected to SDS-PAGE, transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Co., Bedford, MA, USA). Membranes were incubated with primary antibody at a concentration of 2 μg/ml, overnight at 4°C. Abs against phospho-NF-κB p65 (Cat# 3031), NF-κB p65 (Cat# 8242), phospho-Stat1 (Tyr701) (Cat# 9171), phospho-Jak2 (Cat# 3771), Stat1 (Cat# 9172) and Jak2 (Cat# 3230) were obtained from Cell Signaling Technology (CST, USA). Ab against iNOS (Cat# ab15323) was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit IgG (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., China) was added at a concentration of 0.8 μg/ml to the membranes for 1 h at room temperature. The membranes were then developed by ECL using SuperSignal West Pico Trial Kit (Pierce Biotechnology, Rockford, USA).
5
Western Blot Analysis of HO-1 in Murine DCs
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Murine DCs extracts were boiled, electrophoresed on a sodium dodecyl sulfate polyacrylamide gel for 30 min at 80 V, followed by 60 min at 110 V. Membranes were blocked in PBS containing 5% BSA and, after washing, incubated overnight with rabbit anti-HO-1 polyclonal Ab in PBS containing 0.5% Tween-20. After washing, the membranes were incubated for 2.5 h with HRP-conjugated goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology Co., Ltd., China; 1:8,000). Tubulin was used as an intrinsic quality control. The specific bands were visualized using the ECL reagent (Beyotime, Beijing, China) and recorded by a Molecular Imager ChemiDoc XRS System (Bio-Rad, USA).
6
Immunohistochemical Analysis of Bone Markers
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The sections were incubated at 60°C overnight and deparaffinized. Antigen retrieval was performed with 0.1% trypsin for 20 min, and the sections were incubated with 3% H2O2 for 10 min, blocked with goat serum for 10 min, and treated at 37°C for 75 min with rabbit anti-human β-catenin (1:100; ab32572) or anti-human sclerostin (1:50; ab63097) (both from Abcam, Cambridge, MA, USA) monoclonal antibodies in phosphate-buffered saline containing Tween-20 (PBST). Following 3 washes with PBST (5 min), the sections were incubated with HRP-conjugated goat anti-rabbit IgG (1:2,000; ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) at 37°C for 40 min. Visualization was carried out with 3,3′-diaminobenzidine (DAB), and hematoxylin used for counterstaining. Routine dehydration and mounting were performed. In the negative controls, the primary antibody was replaced with PBST. For histological scoring, 3 high power fields (magnification, ×20) were randomly selected from the subchondral bone plate and trabecular bone sections. This was performed independently by 2 experienced pathologists (Dr Anshan Han and Dr Haiping Tian; Ningxia Medical University). The numbers of β-catenin- or sclerostin-positive cells were counted, and positive-to-total cell ratios were considered to be the relative protein expression levels.
7
Immunohistochemical Analysis of LC3A/B in Mammary Tissues
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The mammary tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 4-μm. The sections were rehydrated, treated with citrate buffer (10 mM, pH6) to exposure antigen, and incubated with 3% H2O2 for 30 min to eliminate peroxidase. After washing with PBS, the sections were blocked with 5% bovine serum albumin and incubated with rabbit polyclonal anti-LC3A/B (1:100 dilution, 12741) (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After washing with PBS, the sections were incubated with HRP-conjugated goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) at room temperature for 1 h and then were visualized with DAB Detection Kit (Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Negative controls were performed using the same procedure with the exception of replacing the primary antibody with PBS and irrelevant rabbit serum in each batch. Images were captured using an Olympus BX41 microscope (Olympus, Tokyo, Japan).
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