For HPLC analysis, esculin (EC, purity 98%) and esculetin (ECT, purity 98%) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Fraxin (FR, purity 98.9%) and formic acid (analytical reagent grade) were purchased from Merck KGaA (Darmstadt, Germany). For animal experiments, reserpine (purity 98%), esculin (purity 98%), esculetin (purity 98%), and fluoxetine (FXT, purity 98% in thin layer chromatography) were supplied by Sigma-Aldrich. Fraxin (purity 98%) was supplied by InterPharm Corporation (Koyang-si, Gyeonggi-do, South Korea). For anesthesia, tiletamine/zolazepam was supplied by Virbac (Zoletil 50; Cedex, France). For western blot analysis, actin antibody was supplied by Sigma- Aldrich. BDNF antibody was supplied by Abcam plc. (Cambridge, United Kingdom). CREB antibody and phosphorylated CREB (pCREB) antibody were supplied by Cell Signaling Technology (Danvers, MA, United STates). For immunofluorescence analysis, BDNF antibody was supplied by Abcam plc, pCREB antibody by Cell Signaling Technology, and NeuN antibody by Merck KGaA.
Bdnf antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The BDNF antibody is a research tool used to detect and quantify brain-derived neurotrophic factor (BDNF), a protein involved in the growth, development, and survival of neurons. This antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and localization of BDNF in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti gapdh antibody, by Wuhan Servicebio Technology (2 mentions)
Neun antibody, by Merck Group (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
Pcreb antibody, by Cell Signaling Technology (1 mentions)
Esculin, by Merck Group (1 mentions)
Esculetin, by Merck Group (1 mentions)
Reserpine, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Creb antibody, by Cell Signaling Technology (1 mentions)
Ne per extraction kit, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti hmox 1 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti, by Abcam (1 mentions)
Diaminobenzidine dab, by ZSGB-BIO (1 mentions)
Eclipse ti confocal microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Cryotome e, by Thermo Fisher Scientific (1 mentions)
Eclipse ti s microscope, by Nikon (1 mentions)
Arc antibody, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Cell Signaling Technology (1 mentions)
10 protocols using bdnf antibody
1
HPLC Analysis of Bioactive Compounds
2
Protein Fractionation and Western Blot Analysis in Brain Tissues
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Nuclear and cytoplasmic protein fractions were isolated from frozen frontal cerebrum, hippocampus, amygdala, and cerebellum samples by NE-PER Extraction Kit (Thermo Scientific, Cat. No. 78833), according to the manufacturer’s instruction and as described previously (Olson et al., 2014 (link), 2018 (link)). Western blot experiments were performed with the described protocols in previous reports (Rastegar et al., 2000 (link); Wu et al., 2001 (link); Gordon et al., 2003 (link); Sheikholeslami et al., 2019 (link)). As housekeeping protein and loading control, GAPDH was checked on all membranes and the results for each antibody were normalized to GAPDH signals. Validation of GAPDH in the nuclear and cytoplasmic extracts from human RTT and control brain tissues has been also reported recently (Olson et al., 2018 (link)). The list of primary and secondary antibodies is provided in Supplementary Table S2 . MeCP2E1 and MeCP2E2 antibodies are custom-made and have been previously characterized and reported for detecting endogenous protein isoforms (Zachariah et al., 2012 (link); Liyanage et al., 2013 (link); Olson et al., 2014 (link); Yasui et al., 2014 (link)). BDNF antibody was purchased from Abcam (108319).
3
Immunohistochemical Analysis of p-CREB and BDNF
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After rehydration, the brain tissue sections were immersed in 3% H2O2 solution for 30 min at room temperature to block endogenous peroxidase activity. The tissue sections were then subjected to citrate buffer (10 mM, pH 6.0) for antigen retrieval in a microwave oven. Normal goat serum was added for another 30 minutes at room temperature to block nonspecific binding, followed by incubation with rabbit anti phosphorylated CREB (p-CREB) antibody (1:200, CST, USA) or rabbit recombinant anti-brain-derived neurotrophic factor (BDNF) antibody (1:400, Abcam, Cambridge) overnight at 4°C. After washing with PBS for 15 min, the tissue sections were incubated with the secondary antibody for 1 hour. The excess secondary antibody was then removed and a streptavidin peroxidase complex was placed on the tissue sections for 20 min, followed by 3,3-diaminobenzidine (DAB) for several minutes for dyeing. The chromogenic condition was observed under the microscope and the chromogenic time was recorded while the target protein was brownish-yellow. The tissue sections were then counterstained with hematoxylin, differentiated with alcohol and hydrochloric acid, and flushed with water. No brown structures were observed in the blank control sections. Image J analysis software was used for quantification.
4
Antioxidant and Anti-inflammatory Effects of XYW
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The XYW (Tai Ji, China, batch number 1707029) and fluoxetine hydrochloride (FLX) (Patheon, France, 7686 A) were dissolved in pure water solution preparation (SOD, batch number 20180,309), malondialdehyde (MDA, batch number 20180,313) (GSH, batch number 20180,621), and GSH/glutathione disulfide (GSH/GSSG, batch number 20180,726) levels were assessed using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Nitric oxide (NO) assay kit (Biyuntian, 0,72117,171,110, China). Rat interleukin 6 (IL6) ELISA Kit (MULTI SCIENCES, A30680231), rat interleukin 1β (IL1B) ELISA Kit (ExCell Bio, 21H183), rabbit anti-NTRK2 antibody (Cell Signaling Technology; Cat. No. #4603), rabbit anti-AKT1 antibody (Cell Signaling Technology; Cat. No. #4691S), rabbit anti-p-AKT1 antibody (Cell Signaling Technology; Cat. No. #4060S), rabbit anti-HMOX1 antibody (Cell Signaling Technology; Cat. No. #S2206S), rabbit anti-PIK3CA antibody (Servicebio; Cat. No. #LS190932), rabbit anti-GAPDH antibody (Servicebio; Cat. No. #GB11002), rabbit anti-SOD1 antibody (Novus Biologicals; Cat. No. #03253462C2B), rabbit anti-NFE2L2 antibody (Invitrogen; Cat. No. #TK2668681A), rabbit anti-brain-derived neurotrophic factor (BDNF) antibody (Abcam; Cat. No. #ab108319) and anti-rabbit IgG HRP-linked antibody (Servicebio; Cat. No. #GB23303).
5
Immunohistochemical Analysis of Microwave-Exposed Brain
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Paraffin-embedded brain sections of each group at 7d after microwave exposure were used for immunohistochemistry: (1) AchE was detected by mouse sourced AchE antibody (Novus Biologicals, Colorado,USA); (2) BDNF was detected by rabbit sourced BDNF antibody (Abcam, Cambridge, UK); (3) COX was detected by rabbit sourced COX antibody (Cell Signaling Technology, Massachusetts, USA); (4) SOD was detected by rabbit sourced SOD antibody (Cell Signaling Technology, Massachusetts, USA). Then, the sections were treated with horseradish peroxidase conjugated goat sourced mouse or rabbit antibody. Tissue sections were then colorized with diaminobenzidine (DAB) (ZSGB, Beijing, China). The positive tissue sections were analyzed with CMIAS pathological image analysis system (Beijing University of Aeronautics and Astronautics, China).
6
Immunofluorescence Analysis of Hippocampal Proteins
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Immunofluorescence was performed in order to determine the expression of Arc, BDNF and Reelin in the hippocampus, as described in our previous studies (Ni et al., 2015 (link)). The rat hippocampus was fixed with 4% paraformaldehyde for 24 h, cryoprotected with 30% sucrose for 48 h, and sectioned using a cryostat (Cryotome E, Thermo Fisher, MA, United States). Coronal sections (10 μm thickness) were incubated with Arc antibody (1:200 dilution; Abcam, Cambrige, United Kingdom), BDNF antibody (1:1000 dilution; Abcam, Cambrige, United Kingdom) or Reelin antibody (1:500 dilution; Abcam, Cambrige, United Kingdom) overnight at 4°C, followed by incubation with goat anti-rabbit IgG conjugated to Alexa Fluor® 488 (1:400 dilution; Servicebio, Wuhan, China) for 50 min at room temperature. Nuclei were subsequently counterstained with DAPI (1:5000 dilution; Servicebio, Wuhan, China) for 10 min at room temperature. Images were captured using a Nikon Eclipse Ti confocal microscope. Due to the results of previous studies (Lee and Kesner, 2002 (link)), the hippocampal subregions CA1 and DG serve important roles in retrieving contextual memory after a long time period (i.e., 24 h), these two regions were analyzed for Arc, BDNF and Reelin expressions. Images were acquired by Nikon Eclipse Ti-S microscope (Nikon Corp., Tokyo, Japan).
7
Western Blot Analysis of BDNF Protein
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Protein lysates were extracted from lesioned sciatic nerve tissues or cell cultures through direct homogenization, and lysis in a Laemmli sample buffer (2% SDS, 52.5 mM Tris-HCl, and protein inhibitors). The protein concentration was determined by the Micro BCA Protein Assay Kit (Pierce, Rockford, IL). Protein lysates were mixed with β-mercaptoethanol, glycerin, and bromophenol-blue, and allowed to incubate at 95 °C for 5 min. Equal amounts of protein were separated on 12% SDS-polyacrylamide gels. Following electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Red, Hercules, CA). Membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween-20 for 2 h, probed with primary BDNF antibody (Abcam, Cambridge, MA) overnight at 4 °C, incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce), developed with enhanced chemiluminescence reagent (Cell Signaling, Beverly, MA), and exposed to Kodak X-Omat Blue Film (NEN life science, Boston, MA). Quantification of band signal intensity was conducted with Grab-it 2.5 and Gelwork software.
8
Molecular Mechanisms of Antidepressant Effects
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Xiaoyao Pills (XYW) (TaiJi, China), fluoxetine hydrochloride (FLX) (Patheon, France) and Amitriptyline hydrochloride (Sigma, USA) were dissolved in 0.9% saline to prepare solutions. Paeoniflorin (C23H28O11), Liquiritin (C21H22O9), Glycyrrhizic acid (C42H62O16), Ligustilide (C12H14O2) (Cheng Du Ai Fa, China). LPS (Escherichia coli 055:B5) was provided by Sigma (St. Louis, MO). Rabbit anti-TrkB antibody (1:1000; Cell Signaling Technology; Cat. No. #4603), rabbit anti-cAMP response element-binding protein (CREB) antibody (1:1000; Cell Signaling Technology; Cat. No. #9197S), rabbit anti-p-CREB antibody (1:1000; Cell Signaling Technology; Cat. No. #9198S), rabbit anti-β-Tubulin antibody (1:1000; Cell Signaling Technology; Cat. No. #2128). Rabbit anti-brain-derived neurotrophic factor (BDNF) antibody (1:1000; Abcam; Cat. No. #ab108319). Rabbit anti-DLG4, PSD95-specific, polyclonal antibody (1:1000; Proteintech; Cat. No. #20665-1-AP) and rabbit anti-synaptophysin polyclonal antibody (1:1000; Proteintech; Cat. No. #17785-1-AP). Rabbit anti-GAPDH antibody (1:1000; Servicebio; Cat. No. #GB11002). Rabbit anti-IL-6 polyclonal antibody (1:300; Servicebio; Cat. No. #GB11117) and Alexa Fluor 488 goat anti-rabbit IgG (1:400; Servicebio; Cat. No. #GB25303).
9
BDNF Protein Expression Analysis
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Forty-eight hours after cell transfection, the total protein was extracted, and the concentration was measured by a BCA protein assay kit (Pierce, Rockford, IL, United States). After denaturation at 95°C for 5 min, equal amounts of protein (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and then transferred into the polyvinylidene fluoride (PVDF) membranes (Millipore, United States). Membranes were incubated with BDNF antibody (Abcam, Cambridge, MA, United States) overnight at 4°C, and then probed with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, United States). After being visualized using an enhanced chemiluminescence reagent (Millipore, Billerica, United States), the relative intensities of protein bands were quantified and normalized to GAPDH using ImageJ software (NIH, United States).
10
Immunohistochemical Analysis of BDNF and Amyloid-β
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Dried hippocampal tissue sections were separately taken to be deparaffinized and washed with water, then heat repaired and treated with 3% hydrogen peroxide deionized water. The treated sections were incubated with primary and secondary antibodies respectively. Diaminobenzidine method (DAB) was used for color development, and finally hematoxylin was used for re-staining and mounting the film for observation. Anti-mouse/rabbit two-step detection kit (Ebiogo, 11162110), BDNF antibody (Abcam GR3227037-1), and Aβ antibody (Abcam GR3235744-9) were used.
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