Total protein samples were extracted from the cortex and hippocampus using RIPA lysis buffer. Protein concentrations were detected by using a BCA Protein Assay Kit (Beyotime, China). Proteins were separated and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% bovine serum albumin (BSA), the membrane was incubated overnight at 4°C with the primary antibody. Subsequently, the HRP-conjugated secondary antibodies (1: 1000, Proteintech, China) were incubated with the membranes for 1 h at room temperature. Finally, the levels of target proteins were visualized with a gel imaging apparatus (Bio–Rad, USA). After three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1: 1000, Proteintech, China) for 1 hour at RT and were then washed with TBST an additional 3 times. Finally, the expression of proteins was visualized with a gel imaging apparatus (Bio–Rad, USA). The primary antibodies used were as follows: antibody against COX-2 (1: 1000, Abcam, UK), antibody against DP1 (1: 1000, Abcam, UK), antibody against DP2 (1: 1000, Santa, UK), antibody against caspase-3 (1: 1000, Abcam, UK), antibody against cleaved caspase-3 (1: 1000, Cell Signaling Technology, UK), antibody against caspase-9 (1: 1000, Santa, USA), antibody against Bcl-2 (1: 2000, Abcam, UK), and β-actin (1: 3000, Proteintech, USA).
Hrp conjugated secondary antibody
Manufactured by Proteintech
Sourced in United States, China, Germany
HRP-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassay techniques, such as ELISA and Western blotting. These antibodies are designed to bind to primary antibodies and are conjugated with the enzyme horseradish peroxidase (HRP). The HRP enzyme can catalyze a chromogenic or chemiluminescent reaction, which enables the detection and quantification of target analytes in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (51 mentions)
Ripa lysis buffer, by Beyotime (22 mentions)
Ripa buffer, by Beyotime (17 mentions)
Bca protein assay kit, by Beyotime (16 mentions)
Gapdh, by Proteintech (14 mentions)
β actin, by Proteintech (13 mentions)
Polyvinylidene fluoride membrane, by Merck Group (9 mentions)
Polyvinylidene difluoride membrane, by Merck Group (9 mentions)
Bca kit, by Beyotime (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ecl kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor, by Beyotime (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ecl reagent, by Thermo Fisher Scientific (6 mentions)
Anti gapdh, by Proteintech (5 mentions)
Nitrocellulose membrane, by Merck Group (5 mentions)
Anti β actin, by Proteintech (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Bca assay, by Beyotime (4 mentions)
180 protocols using hrp conjugated secondary antibody
1
Western Blot Analysis of Protein Expression
2
Quantifying DNA Damage in Kidney Tissue
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After 4-µm kidney sections were deparaffinized and hydrated, each section was exposed to 3% hydrogen peroxide solution. After antigen repair, kidney sections were incubated with primary antibodies against γ H2AX (1:200, Abcam, ab2893) overnight at 4°C. Subsequently, sections were incubated with HRP-conjugated secondary antibody (Proteintech Group, Inc., Chicago, IL, United States) at room temperature for 30 min; a DAB kit (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was used for localization. Images were taken through a microscope (×400).
3
Quantitative Protein Expression Analysis
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The protein samples were harvested using a total protein lysates kit (cat no. KGP250; Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). The protein concentrations were determined using the BCA Protein Assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) according to the manufacturer’s instructions. A total of 25 µg protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis (gel, 10%; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to a PVDF membrane (Immobilon; EMD Millipore, Bedford, MA, USA). Immunoprobing was performed by incubating the membrane with anti-GAPDH antibody (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-CTSB antibody (1:1,000; cat. no. ab230401, Abcam), anti-FAK antibody (1:1,000; cat. no. WL01696; Wanleibio Co., Ltd., Shanghai, China) and anti-MMP-9 antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4°C, followed by binding with a HRP-conjugated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used for detection.
4
Isolation and Identification of Diterpenes
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The four diterpenes, including ajudecumin A (1 ), Ajuforrestin B (2 ), (16S)-12,16-epoxy-11,14-dihydroxy-17(1 5 →16)-abeo-abieta-8,11,13-trien-7-one (3 ), and 14,15-dihydroajugapitin (4 ) were isolated from Ajuga ovalifolia var. calantha and identified by NMR (Chen et al. 2017b ), (Supplementary Figures S1 and S2 ). LPS from Escherichia coli 055:B5 and carrageenan were provided by Sigma (Shanghai, China). BAY 11-7082, CCK-8 agent, Griess agent was obtained from Beyotime (Haimen, China). Antibody against iNOS is the products of Cell Signalling Technology (Danvers, USA). Phospho- and total-ERK, phosphor- and total-p38 MAPK, phosphor- and total-JNK, phosphor- and total-IκBα antibodies were purchased from Signalway Antibody (Baltimore, USA). COX-2 and actin antibodies as well as HRP-conjugated secondary antibody were from Proteintech (Wuhan, China).
5
Western Blot Analysis of LepRb in Hippocampus and Hypothalamus
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Hippocampus and hypothalamus (30 mg, separately) were homogenized on ice with 2 mM phenylmethanesulfonyl fluoride in 1 ml ice-cold RIPA buffer added protease inhibitor. The protein concentration in the supernatant was harvested and determined by the BCA analysis. Equal amounts of protein (40 ug) from each sample were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Boston, MA). The membranes were blocked by non-fat dry milk buffer for 1 hour and then immunoblotted overnight at 4 °C with primary antibody against LepRb (sc-8325, Santa Cruz Biotechnology, dilution 1:250) and β-actin (Proteintech, USA; dilution 1:1000). After being washed for three times, the membranes were incubated with HRP-conjugated secondary antibody (Proteintech, USA; dilution 1:2000) for 1 hour at room temperature, washed and visualized by employing enhanced chemiluminescence. The optical densities of bands were recorded and analyzed by using C-Digit (LI-COR, USA). Relative expression was normalized to β-actin.
6
Western Blotting Analysis of Ischemic Brain
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After treatment, cells and cortex tissues of ischemic brain were harvested and lysed by radio-immunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (PMSF) on ice; they were then centrifuged at 12,000× g for 15 min at 4 °C. Then, equal amounts of proteins were electrophoresed on 12% density sodium dodecyl sulfate, sodium salt (SDS) acrylamide gels, transferred to a polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk (Cell Signaling Technology, Danvers, MA, USA) in tris-buffered saline and tween 20 (TBST) for 2 h, followed by overnight incubation with antibodies to CREB (1:1000), p-CREB (1:1000), CaMKII (1:1000), p-CaMKII (1:1000), CaM (1:500) and β-actin (1:1000). Membranes were then incubated for 2 h at room temperature with HRP-conjugated secondary antibody (Proteintech Group, Chicago, IL, USA, 1:7000). Finally, all specific bands were visualized using the electrochemical luminescence (ECL) western detection reagents on Image Lab analysis software (Bio-Rad, Philadelphia, PA, USA). β-actin was used as a loading control. Three repeats of the experiments were performed.
7
Western Blot Analysis of Endothelial Markers
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Protein lysate samples were boiled at 95°C, run on 4-15% polyacrylamide gels, and electrophoretically transferred onto PDVF membranes. Membranes were blocked with 5% nonfat milk/TBST for 1 h and incubated overnight at 4°C with the following primary antibodies: VE-cadherin (Santa Cruz, 1 : 500), Zo-1 (Invitrogen, 1 : 500), beta-actin (Cell Signaling Technology, 1 : 1000), Tecr (Bethyl laboratories, 1 : 1000), Cav-1 (NOVUS, 1 : 1000), and Claudin-5 (Thermo Fisher Scientific, 1 : 1000). Membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Proteintech, 1 : 5000) and developed with regular ECL (Thermo Fisher Scientific). The intensity of individual bands was quantified using ImageJ.
8
Western Blot Analysis of Autophagy and Inflammation
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Protein samples were boiled for 5 min in 5× SDS-PAGE loading buffer and loaded onto 10–15% SDS polyacrylamide gels. Proteins were transferred to PVDF membranes, and membranes were blocked for 2 h in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 with 5% non-fat dry milk and then incubated at 4° C overnight with the following primary antibodies: anti-LC3B (1:2,000; Sigma-Aldrich), anti-P62 (1:5,000; Proteintech), anti-P65 (1:1,000; Cell Signaling Technology), anti-phospho-P65 (1:1,000; Cell Signaling Technology), anti-ICAM-1 (1:200; Santa Cruz Biotechnology), anti-PI3K (1:1,000; Cell Signaling Technology), anti-phospho-PI3K (1:1,000; Cell Signaling Technology), anti-Akt (1:1,000; Cell Signaling Technology), anti-phospho-Akt (1:1,000; Cell Signaling Technology), anti-phospho-p70S6K (1:1,000; Cell Signaling Technology), or anti-GAPDH(1:50,000; Proteintech). Blots were washed and then incubated with the HRP-conjugated secondary antibody (1:10,000; Proteintech), and blots were revealed using ECL. ImageJ software was used for the semi-quantification of protein expression.
9
Quantitative Protein Analysis via SQLE
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Total proteins were extracted in RIPA buffer, and the protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Protein was resolved in 10% Tris-SDS-PAGE gels and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were incubated with anti-human SQLE antibody (Proteintech, Chicago, IL) at a dilution of 1:1000 and then probed with HRP-conjugated secondary antibody (Proteintech, Chicago, IL).
10
Western Blot Analysis of FPR3 and CCR2 Proteins
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Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) in the presence of Cocktail protease inhibitor (Abmole, USA) and Cocktail phophatase inhibitor (Abmole, USA). Lysate protein concentrations were determined by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equivalent protein from different samples was separated by 10% SDS-PAGE protein electrophoresis and the separated protein samples were transferred onto the Immobilon PVDF membranes. Then, the membranes were incubated with 5% skim milk to block the non-specific sites. Rabbit polyclonal anti-human FPR3 antibody (orb608040, Biorbyt, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337, Novus Biologicals, USA) and HRP-conjugated secondary antibody (1:2000, Proteintech, IL, USA) were used. GAPDH (1:5000, Proteintech, IL, USA) was simultaneously used as an internal control. Signals were detected and visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany).
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