The cortex and cerebral microvessels of right hemisphere were separated for western blot analysis. Samples were incubated with protease inhibitor (Roche) then diluted with sample buffer. Samples were supplemented with 2% beta-mercaptoethanol and 50 mM DTT and boiled for about 5 min. Proteins were separated on tris-glycine 4–15% acrylamide gels and transferred to PVDF membranes soaked in 5% nonfat milk in PBS-Tween 20 (0.05%) for 2 h. The proteins of total caspase-3, cleaved caspase-3, Ser1177 phosphorylated endothelial nitric oxide synthase (Ser1177 p-eNOS) and β-actin were tested by rabbit monoclonal antibody against caspase-3 (Santa Cruz, 1:500), rabbit polyclonal antibody against cleaved caspase-3 (Biorbyt, 1:500), rabbit polyclonal antibody against Ser1177 p-eNOS (Invitrogen, 1:400) and rabbit polyclonal antibody against β-actin (Santa Cruz, 1:500) respectively. Immunoreactivity was tested by incubation with secondary HRP-coupled antibody for 1 h at room temperature followed by the ECL plus reagent (Santa Cruz). The densities of the bands were determined by the MiVnt image analysis system (Bio-Rad, Carlsbad, CA, USA).
Mivnt image analysis system
Manufactured by Bio-Rad
Sourced in United States
The MiVnt image analysis system is a compact and versatile lab equipment designed for performing advanced image analysis tasks. It provides a robust platform for capturing, processing, and analyzing a wide range of digital images and microscopic samples. The MiVnt system offers a user-friendly interface and powerful software capabilities to support various research and analytical applications.
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Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
Rabbit polyclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Ecl plus reagent, by Santa Cruz Biotechnology (1 mentions)
Caspase 1 p20, by Adipogen (1 mentions)
Caspase 1, by Abcam (1 mentions)
Hypersensitivity chemiluminescent substrate, by Bio-Rad (1 mentions)
Nlrp3, by Abcam (1 mentions)
Image lab v5, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti proliferating cell nuclear antigen, by Abcam (1 mentions)
Goat anti homer1a, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti psd95, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
4 protocols using mivnt image analysis system
1
Western Blotting of Cortical Proteins
2
Western Blot Analysis of NLRP3 Inflammasome
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We used lysis buffer to lyse the spinal cord tissue and primary cultured neurons. After protein quantification, electrophoresis, and Western transfer, we blocked the membranes with 5% bovine serum albumin followed by incubation with the appropriate primary antibodies as follows: NLRP3 (Abcam, 1:1,000), Caspase-1 (Abcam, 1:1,000), Caspase-1 (p20) (AdipoGen, 1:1,000), ASC (Abcam, 1:1,000), LC3 (CST, 1:2,000), and Beclin 1 (CST, 1:1,000), at 4°C overnight. Next, we incubated the membranes with secondary antibodies (Abgent, 1:30,000) followed by the hypersensitivity chemiluminescent substrate (Bio-Rad, United States). We obtained images using a MiVnt image analysis system (Bio-Rad, United States).
3
Protein Quantification and Western Blot Analysis
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Total proteins were obtained from the PASMCs of lung tissue samples and cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were quantified using a bicinchoninic acid protein kit (Invitrogen; Thermo Fisher Scientific, Inc.). Protein (40 μg/lane) was loaded onto 10% SDS-PAGE gels. Following SDS-PAGE and transfer to a PVDF membrane (EMD Millipore), membranes were blocked in 5% BSA (Sigma-Aldrich; Merck KGaA). Membranes were incubated with the primary antibodies at 4°C overnight. The primary antibodies used were anti-NOR1 (cat. no. ab94507; 1:1,000; Abcam), anti-proliferating cell nuclear antigen (PCNA; cat. no. ab92552; 1:1,000; Abcam) and anti-GAPDH (cat. no. 5174S; 1:2,000; CST Biological Reagents Co., Ltd.). Subsequently, membranes were incubated with the Horseradish peroxidase, Goat Anti-Rabbit IgG secondary antibody (cat. no. A21020; 1:20,000; Abbkine Scientific Co., Ltd.) for 2 h at room temperature. Signals were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.) and detected using a MiVnt image analysis system (Bio-Rad Laboratories, Inc.). Optical densities of the bands were calculated using Image Lab v5.2.1 (Bio-Rad Laboratories, Inc.).
4
Western Blot Analysis of Neuronal Proteins
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WB analysis was performed as previously described.61 (link) After various treatments, proteins were extracted, quantified and separated by electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk. After blocking, the membranes were incubated with the appropriate primary antibodies: goat anti-Homer1a (1 : 200; Santa Cruz, Dallas, TX, USA), mouse anti-Homer1b/c (1 : 1000; Santa Cruz), rabbit anti-PSD95 (1 : 1000; Santa Cruz), rabbit anti-p-Erk, Erk, NR1, NR2B, p-nNOS, nNOS, CREB, p-CREB (1 : 1000; Cell Signaling Technology (CST), Danvers, MA, USA) and mouse anti-β-Actin (1 : 2000; Sigma-Aldrich). Immunoreactivity was detected by incubation with horseradish peroxidase-conjugated horse anti-secondary antibodies (1 : 20000; CST) followed by chemiluminescent substrate development (Thermo Fisher Scientific Inc., Waltham, MA, USA). WBs for each protein were repeated three times and the optical densities of the bands were calculated using a MiVnt image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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