Quantitative measurement of MMP-9, D1R, and Cav1.2 in such rat brain structures as the vSTR was performed using a Rat MMP-9 ELISA Kit (Reddot Biotech, Kelowna, BC, Canada), a Rat Dopamine Receptor D1 (DRD1) ELISA kit (Reddot Biotech, Kelowna, BC, Canada), and Rat Voltage-dependent L-type Calcium Channel Subunit Alpha-1C ELISA Kit (Bioassay Technology Laboratory, Shanghai, China), respectively, following manufacturers’ protocols. Firstly, frozen brain structures were homogenized in cold buffer at pH 7.4 (0.32 M sucrose, 1 mM HEPES, 1 mM MgCl2, 1 mM NaHCO3, and 0.1 mM PMSF) containing cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, MO, USA), using a homogenizer ball (Bioprep-24, Allsheng, China) (10 s at 10,000 rpm). Then, homogenates were centrifuged for 5 min at 5000× g, the supernates were immediately removed, and protein concentration in the supernates was measured using a bicinchoninic acid assay (BCA) protein assay kit (Serva, Heidelberg, Germany). From each sample, 100 µg of protein was used in each ELISA assay. All data are expressed in ng/mL.
Bioprep 24
Manufactured by Allsheng
Sourced in China
The Bioprep-24 is a high-performance tissue homogenizer designed for rapid and efficient sample preparation. It features 24 sample tubes that can be processed simultaneously, ensuring consistent and reproducible results. The Bioprep-24 utilizes advanced technology to thoroughly disrupt and homogenize a wide range of sample types, including tissues, cells, and other biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Cocktails of protease and phosphatase inhibitors, by Merck Group (1 mentions)
Bca protein assay kit, by Serva Electrophoresis (1 mentions)
Accupower rt premix, by Bioneer (1 mentions)
Exicycler tm 96 real time quantitative pcr system, by Bioneer (1 mentions)
Nanodrop spectrophotometer, by DeNovix (1 mentions)
Accupower greenstar qpcr premix, by Bioneer (1 mentions)
Ab110334, by Abcam (1 mentions)
Ab89295, by Abcam (1 mentions)
Chemiluminescence kit, by Bio-Rad (1 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Abs204, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Goat anti rabbit secondary antibody, by LI COR (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
5 protocols using bioprep 24
1
Quantitative Analysis of MMP-9, D1R, and Cav1.2 in Rat Brain
2
Liver Tissue Analysis: Gene Expression
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Livers of rats were taken and divided. One of the samples of livers were stored in formaldehyde for TUNEL staining, and another of the samples of livers were stored at -86°C until further analysis. Thirty mg of frozen liver tissues were homogenized in 500 µl Tissue Lizis Buffer for 1 min using homogenizer (Bioprep-24, Allsheng). Total RNA was obtained from liver samples using an ExiPrepTM Tissue Total RNA isolation kit (Bioneer, K-3325). The RNA concentration was determined from absorbance at 230-260 nm and 260/280 nm using a NanoDrop spectrophotometer (Denovix DS-11). The results were then reversely transcribed into cDNA using the AccuPower® RT PreMix (Bioneer, K-2041) according to the manufacturer's instructions.
Real-Time PCR was performed using AccuPower GreenStar qPCR PreMix according to the manufacturer's instructions (Bioneer, . The level of mRNA expression of Fas genes as detected using the ExiCyclerTM96 Real-Time Quantitative PCR system (Bioneer). The PCR reactions were performed as follows: 95°C for 5 min, followed by 45 cycles at 95°C for 15 s, and then 60°C for 25 s. The sequences primers used were: Forward, 5'-AGGTGCTA-GAGGCCCTGCTA-3'; Reverse, 5'-GTGCACAGACAC-CTTCCCAT-3' (Bioneer, S-1001) (Ji et al. 2011; (link)Fukunishi et al. 2014) (link). The levels of each gene expression were calculated by the 2 -ΔΔCt method.
Real-Time PCR was performed using AccuPower GreenStar qPCR PreMix according to the manufacturer's instructions (Bioneer, . The level of mRNA expression of Fas genes as detected using the ExiCyclerTM96 Real-Time Quantitative PCR system (Bioneer). The PCR reactions were performed as follows: 95°C for 5 min, followed by 45 cycles at 95°C for 15 s, and then 60°C for 25 s. The sequences primers used were: Forward, 5'-AGGTGCTA-GAGGCCCTGCTA-3'; Reverse, 5'-GTGCACAGACAC-CTTCCCAT-3' (Bioneer, S-1001) (Ji et al. 2011; (link)Fukunishi et al. 2014) (link). The levels of each gene expression were calculated by the 2 -ΔΔCt method.
3
Quantifying Hippocampal Biomarkers in Rats
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Rat hippocampal tissues were mixed with a volume of normal saline 10 times the tissue mass. Then, hippocampal tissues were homogenized in a Bioprep-24 tissue homogenizer (Allsheng, Hangzhou, Zhejiang, China) and centrifuged (4000 rpm) to collect the supernatants. The levels of the brain biomarkers brain-derived neurotrophic factor (BDNF), monocyte chemoattractant protein-1 (MCP-1), and homocysteine (Hcy) were measured in the supernatant samples or cell supernatants according to the manufacturer’s instructions. All results were obtained by measuring the optical density using a microplate reader. The BDNF (cat. no. EK0308) ELISA kit was purchased from Boster Bioengineering Company (Wuhan, China). The MCP-1 (cat. no. SBJ-R0616) and Hcy (cat. no. SBJ-R0319) ELISA kits were purchased from SenBeiJia Biological Technology (Nanjing, China).
4
Quantitative Analysis of Phospho-PDH and PDK4 in Heart Tissue
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Heart tissue samples were homogenized with a Bioprep-24 (Hangzhou Allsheng Instruments Co., Ltd.) in T-PER buffer (#78510, Thermo Fisher Scientific, Waltham, MA, USA), protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and phosphatase inhibitor cocktail (Roche Diagnostics). To collect supernatant, tissue lysates were centrifuged at 10,000 rpm for 10 min at 4 °C. Subsequently, 5 μg of protein were loaded in each lane of a 10% Bis–Tris gel (Thermo Fisher Scientific) and separated using SDS-PAGE and electroblotted to nitrocellulose membranes using an iBlot2 Dry Blotting System (Thermo Fisher Scientific). Membranes were blocked with Setsuyakukun supporter (DRC, Tama, Tokyo, Japan) for 1 h at room temperature, washed, and incubated for 2 h at room temperature in Kiwami Setsuyakukun buffer with anti-phospho-PDH antibodies (ABS204 and ABS194, Millipore), anti-total-PDH antibodies (ab110334, Abcam, Cambridge, United Kingdom), anti-PDK4 antibodies (ab89295, Abcam), or anti-GAPDH antibodies (AM3400, Thermo Fisher Scientific). Target proteins were detected with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and a chemiluminescence kit (#170-5060, Bio-Rad Laboratories). Signals were detected with a ChemiDoc Touch (Bio-Rad, Hercules, CA, USA), and quantitative analysis was performed using Image Lab (Bio-Rad).
5
Western Blot Quantification of 5-HT2C Receptors
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Brain structures isolated immediately after behavioral procedures were homogenized in a homogenization buffer (1 mM HEPES, 0.1 M DTT, 0.1 mM EGTA (pH 7.2–7.8), COMPLETE and sterile water) using a homogenizer ball (Bioprep-24, Allsheng, China) at 10 s at 10,000 rpm, and were then denatured for 2 min at 85 °C, 2 min in ice, 5 min at 85 °C, and finally 2 min in ice as described previously by Smaga et al. [61 (link)]. The bicinchoninic acid assay protein assay kit (Serva, Germany) was used for protein determination. Protein samples (40 mg) were resolved in gels and transferred to membrane, and determination of 5-HT2C receptor was performed using anti-5-HT2C rabbit monoclonal antibody (1:1000; Abcam, UK). The expressions of 5-HT2C receptors were evaluated relative to anti-glyceraldehyde 3-phosphate dehydrogenase antibody (GAPDH), using rabbit anti-GAPDH polyclonal antibody (1:1500, sc-25778, Santa Cruz Biotechnology, USA). Blots were washed and incubated with goat anti-rabbit secondary antibody (1:6000; 926–68,071; Li-cor, USA) and visualized with fluorescence detection Odyssey Clx (Licor, USA). The analysis was performed using Image Studio v.2.1. All data were expressed as % of control.
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