7,8 Dihydroxyflavone was purchased from Tocris Bioscience, UK. All other reagents were of analytical grade (Sigma, St. Louis, MO). Anti-BDNF (Santa Cruz, sc-546; 1:1000), anti-pTrkB Y515 (Abcam, ab51187; 1:1000), anti-TrkB (Santa Cruz, sc20542; 1:1000), anti-pAkt (Cell Signalling, 193H12; 1:1000), anti-Akt (Cell Signalling, 11E7; 1:1000), anti-pErk Thr 202/Tyr 204 (Cell Signalling, D12.14.4E; 1:1000), anti-Erk (Cell Signalling 137F5; 1:1000), anti-pGSK3β(Ser9) (Cell Signalling 5B3; 1:1000), anti-GSK3β (Cell Signalling, 27C10; 1:1000) and anti-β-actin (Sigma, AC-40; 1:1000) antibodies were used for Western blotting. Fluorescent polystyrene microspheres from Invitrogen (FluoSpheres; Invitrogen, Carlsbad, CA) were used for anterior chamber injections. All other reagents were of analytical grade (Sigma, St. Louis, MO).
Anti bdnf
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, Italy
Anti-BDNF is a laboratory reagent used to detect and quantify the presence of brain-derived neurotrophic factor (BDNF) in biological samples. It functions as a specific antibody that binds to BDNF, enabling its identification and measurement through various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti trkb, by Santa Cruz Biotechnology (9 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti ngf, by Santa Cruz Biotechnology (3 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Anti erk, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti bax, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Fluosphere, by Thermo Fisher Scientific (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti ptrkb y515, by Abcam (1 mentions)
Anti p erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti p gsk3β ser9, by Cell Signaling Technology (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
42 protocols using anti bdnf
1
Flavonoid-Mediated Neuroprotective Signaling
2
Protein Extraction and Western Blot Analysis from DRG Cells
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Cells were isolated from DRG and made to single cell suspension. Washed with PBS for 2 times, then added RIPA lysis buffer and maintained on ice for 30 min accompanied with vortexing 30 s per 10 min. Centrifuged for 15 min at 4 °C with 12 000 rpm and transferred the supernatant to a new tube. The protein concentrations were measured using Nanodrop 2000 (Thermo Fisher Scientific, San Jose, CA, USA). Then added 10× loading buffer to the protein solution and heated on 95 °C for 5 min. Proteins (10 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA). After blocked with 5% dried skimmed milk in Tris-buffered saline (TBS), the membranes were incubated with specific primary antibodies including anti-BDNF, anti-Wnt5a and anti-β-catenin (1 : 100 dilution, and all from Santa Cruz Biotechnology) and anti-β-actin antibody (1 : 2000 dilution, Santa Cruz Biotechnology) at 4 °C overnight. The PVDF membrane was washed five times with TBS with Tween-20 (TBST), followed by incubation with HRP-conjugated anti-mouse IgG secondary antibody (1 : 2000 dilution, Santa Cruz Biotechnology) at room temperature for 1 h. After being washed with TBST five times, the protein signals were detected using PierceTM ECL western blotting substrate (Thermo Fisher Scientific).
3
Western Blot Analysis of Oxidative Stress Markers
4
Quantifying BDNF Protein in Mouse Hippocampus
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Total proteins in mice hippocampi were extracted using RIPA Lysis Buffer (Beyotime, Guangzhou, China) and the concentration of proteins was determined using the BCA protein assay kit (Pierce, Rockford, USA) according to the manufacturer's instruction. Western blotting assays were performed as previously described (Zhang, Fang, & Wang, 2015 ). Primary antibodies were anti‐BDNF (dilution 1:1,000; #sc‐546, Santa Cruz, Dallas, TX, USA) and anti‐β‐actin (dilution 1:1,000; #sc‐8432, Santa Cruz). The protein expression was visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA). Images were captured using the ChemiDoc XRS imaging system (Bio‐Rad, Hercules, CA, USA) and Quantity One image software was used for the densitometric analysis of each band. β‐actin was used as an internal loading control.
5
Hippocampal Protein Extraction and Analysis
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Protein lysates were extracted according to a previously described method (Hwang et al. 2018 ; Kim et al. 2018 (link)). Dissected hippocampal tissues were homogenized in 400 μl per 1 g concentration of RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM PMSF (Sigma-Aldrich Inc.) on ice. The homogenized sample was incubated on ice for 20 min. Incubated sample was centrifuged at 14,000 g for 10 min at 4°C, and supernatants were collected. Protein content was measured using a micro-drop plate reader (Thermo Fisher Scientific). NF-кB and IкBα in the hippocampus were detected using nuclear/cytosol fractionation kit (BioVision Inc, Milpitas, CA, USA) according to the manufacturer’s instructions. The following primary antibodies (1:1000 dilution) were selected to react overnight at 4°C: mouse anti-β-actin, anti-TNF-α, anti-interleukin-6 (IL-6), anti-proBDNF, anti-BDNF, anti-Iba-1, rabbit anti-TrkB, anti-NF-κB, and anti-IκBα (Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 h with attempt secondary antibodies (1:2000; Vector Laboratories). Blot membrane was detected using the HRP-conjugated IgG (Vector Laboratories) and the enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). Detected bands were quantified by Image-Pro® plus image analysis system (ver. 6.0, Media Cybernetics Inc., Silver Spring, MD, USA).
6
Western Blotting Analysis of Tear Proteins
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Seven micrograms of proteins from tears were subjected to SDS-PAGE (NuPage Novex 4–12% gel; Thermo Fisher Scientific) and then transfer to a PVDF membrane (Bio-Rad). The membranes were blocked with 5% nonfat milk in PBS plus 0.1% Tween 20 and then probed with the specific primary antibodies (rabbit polyclonal anti-BDNF, rabbit polyclonal anti-NGF, and rabbit polyclonal anti-Sema7A from Santa Cruz Biotechnology) all at 1:100 dilution. The membranes were washed with PBS plus 0.1% Tween 20 and further incubated with goat anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology). Protein bands were visualized using chemiluminescent detection reagent (Thermo Fisher Scientific) and quantified with LAS-4000 Imaging System (GE Healthcare, Waukesha, WI, USA).
7
Western Blot Analysis of Hippocampal Proteins
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Western blot was done, as mentioned in the below (Ji et al., 2020 (link)). The hippocampal tissues were homogenized using lysis buffer consisting of 1 mM EGTA, 1 mM PMSF, 1 mM Na2VO4, 1.5 mM MgCl2·6H2O, 50 mM Tris-HCl (pH, 8.0), 100 mM NaF, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and then centrifuged at 50,000 rpm for 1 hr. Anti-β-actin antibody (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-BDNF (1:1,000), anti-TrkB (1:1,000), anti-Bax antibody (1:1,000; Santa Cruz Biotechnology), and anti-Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody for β-actin, Bax, and Bcl-2 (1:3,000; Amersham Pharmacia Biothech GmbH, Freiburg, Germany), and horseradish peroxidase-conjugated anti-rabbit antibody for BDNF and TrkB (1:5,000; Vector Laboratories), were used as the secondary antibodies. In addition to the membrane transfer performed at 4°C, all other steps were performed at room temperature. Band detection was done by enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).
8
Western Blot Analysis of BDNF, PI3K, and AKT
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Tissue samples or cultured cells were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce, Rockford, IL, USA). The total protein concentration was determined by a BCA kit (Pierce). Total protein(30μg) from each sample were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes (Santa Cruz Biotechnology, Inc, USA). After blocked in 5% nonfat milk, the membranes were probed with the primary antibodies overnight at 4°C, and then with the corresponding secondary antibodies conjugated to horseradish peroxidase (HRP, Santa Cruz Biotechnology). Band signals were visualized using an enhanced chemiluminescence kit (Pierce, Minneapolis, MN, USA). Primary antibodies were used as follows: anti-BDNF (1:1000, Santa Cruz Biotechnology), anti-PI3K (1;1000, Cell Signaling Technology, USA),anti-p-PI3K (1;1000, Cell Signaling Technology), anti-AKT (1;1000, Cell Signaling Technology), anti-p-AKT (1:800, Cell Signaling Technology), and anti-GAPDH (1:3000, Santa Cruz Biotechnology). GAPDH was used as an internal control.
9
Brain Tissue Analysis of Ischemic Penumbra
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Six animals in each group were sacrificed 24 h after reperfusion, and the rat brain tissues were extracted from ipsilateral ischemic penumbra. Western blot analysis was performed as described previously 8 (link). Anti-Bax (1:200, Santa Cruz, USA), anti-Bcl-2 (1:200, Santa Cruz, USA), anti-BDNF (1:200, Santa Cruz, USA), anti-cleaved caspase-3 (1:500, Cell Signaling Technology), and anti-GAPDH (1:2000; Santa Cruz, USA) were used as primary antibodies. The blots were subjected to gel formatter (BIO-RAD) and quantified through Quantity One analysis. GAPDH was used as an internal loading control.
10
Wheat AX Antibody Characterization
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Wheat AX was purchased from Megazyme (Wicklow, Ireland). SCO and anti-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-BDNF and anti-phospho-CREB (p-CREB) antibodies were the products of SantaCruz (Santa Cruz, CA, USA) and Cell Signaling (Boston, MA, USA), respectively.
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