Protein was extracted from glioma cells and measured through the BCA kit (Beyotime Biotechnology, China) [18 (link)]. Then, the protein was extracted using SDS-PAGE (10%) and transformed into PVDF membranes (Millipore, USA). Afterwards, membranes were incubated using 5% skimmed milk and incubated with primary antibodies under 4°C overnight. The antibodies are as follows: anti-Bax (1: 2, 000, bs-28034R, Bioss, China), anti-Bcl-2 (1: 2, 000, bs-4563R, Bioss, China), anti-MMP-2 (1: 2, 000, bs-20705R, Bioss, China), anti-MMP-9 (1: 2, 000, bs-22502R, Bioss, China), anti-Cleaved caspase-3 (1: 2, 000, bsm-33199M, Bioss, China), anti-Cleaved caspase-9 (1: 2, 000, bs-3082R, Bioss, China), anti-Cox-2 (1: 2, 000, bs-10411R, Bioss, China), and anti-β-actin (1: 2, 000, bs-0061R, Bioss, China) with β-actin being the endogenous control. Afterwards, membranes were incubated for 1 h using a secondary antibody (1: 2, 000, bs-0311P-HRP, Bioss). Finally, ECL (Millipore, USA) was utilized to observe protein blots and quantified using ImageJ software (version 4.3; National Institutes of Health).
Bs 4563r
Manufactured by Bioss Antibodies
Sourced in China, United States
Bs-4563R is a versatile laboratory equipment designed for diverse applications. It functions as a high-performance centrifuge, capable of separating components from complex mixtures with precision. The core function of this product is to provide efficient and reliable centrifugation for research and analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
Anti mmp 9, by Bioss Antibodies (1 mentions)
Anti β actin, by Bioss Antibodies (1 mentions)
Anti bax, by Bioss Antibodies (1 mentions)
Anti bcl 2, by Bioss Antibodies (1 mentions)
Anti mmp 2, by Bioss Antibodies (1 mentions)
Anti cleaved caspase 3, by Bioss Antibodies (1 mentions)
Anti cox 2, by Bioss Antibodies (1 mentions)
Bs 28034r, by Bioss Antibodies (1 mentions)
Bs 20705r, by Bioss Antibodies (1 mentions)
Bs 10411r, by Bioss Antibodies (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Bs 0127r, by Bioss Antibodies (1 mentions)
Rabbit anti tlr4, by Cell Signaling Technology (1 mentions)
Ab8932, by Abcam (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
4 protocols using bs 4563r
1
Protein Expression Analysis in Glioma Cells
2
Molecular Analysis of Signaling Pathways
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qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-itTM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.
3
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed on the LV sections to evaluate the expression of BAX (LS-C353915, LSBio, Seattle, US), BCl-2 (BS-4563R, Bioss Inc., Boston, US), Caspase3 (SC-56046, Santa Cruz Biotechnology, CA, US), GSK-3β (NBP1-47470, Novus Biologicals, Manama, US), SGK1 (SC-28338, Santa Cruz Biotechnology, CA, US), P-AKT (Biotin, orb501663, Biorbyt, UK), P-ERK1/2 (orb10606, Biorbyt, UK), P-STAT3 (orb704362, Biorbyt, UK) proteins as previously described in detail elsewhere [7 (link)]. GAPDH (ab181602, Abcam, Cambridge, US) was used as the loading control. The intensity of each protein band was semi-quantified by the Image J image analysis system.
4
Immunohistochemical Staining of Brain Tissue After Ischemic Stroke
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Immunohistochemical staining was carried out according to our previous studies38 (link),40 (link). After ischemic stroke, brain sections were stained with hematoxylin–eosin (H.E.) stain and evaluated histologically. Furthermore, coronal brain slices were stained using the following primary antibodies: rabbit anti-BDNF (ab108319; Abcam PLC, Waltham, MA, USA, 1:100); rabbit anti-Bcl-2 (bs-4563R; Bioss Inc., Woburn, MA, USA, 1:200); rabbit anti-Bax (ab32503; Abcam PLC, 1:200); and rabbit anti-caspase 3 (196771-AP; Proteintech Group, Inc., Rosemont, IL, USA, 1:500). These were then deparaffinized, rehydrated, and blocked for 15 min with methanol containing 0.9% hydrogen peroxide for endogenous peroxidase. Then, the sections were washed three times for 5 min each with PBS (pH 7.6) and blocked with 10% skim milk in PBS for 20 min. The sections were then incubated with each of the aforementioned primary antibodies overnight at 4 °C, washed three times for 10 min each in PBS, incubated with peroxidase-conjugated dextran polymer (EnVision; Agilent Dako, Santa Clara, CA, USA) and goat anti-rabbit IgG, and incubated at 25 °C for 60 min. After washing the sections with PBS, immunoreaction was visualized by 3,3′-diaminobenzidine staining.
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