Rabbit anti trkb

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Rabbit anti-TrkB is a primary antibody produced in rabbit that recognizes the TrkB receptor. TrkB is a receptor tyrosine kinase that binds to brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), and plays a role in neuronal development and survival.

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Close spelling variants of this product

Anti-TrkB (16 citations) Rabbit anti-TrkB antibody (4 citations) Rabbit anti-TM (2 citations)

11 protocols using rabbit anti trkb

Immunohistochemical Visualization of Bone Markers

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Immunohistochemistry was performed using the Envision+/HRP system (Dako) with rabbit anti-ALP [22 (link)], rabbit anti-osteopontin (LSL), rabbit anti-trkB (Santa Cruz) and mouse anti-cathepsin K (Daiichi Fine Chemical Co., Ltd, Toyama, Japan) antibodies. Sections were treated with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature to block endogenous peroxidase activity. After rinsing in PBS, the sections were incubated with 5% skimmed milk in PBS and 0.05% Triton X-100 (T-PBS) for 1 hour at room temperature to block non-specific protein-binding sites. They were then incubated overnight at 4°C with the primary antibodies diluted at 50 μg/ml in T-PBS. For visualization of the reaction products, sections were the treated with 0.02% 3,3’-diaminobenzidine (Dohjin Laboratories) in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.005% hydrogen peroxide, and they were counterstained with hematoxylin. For control experiments, the primary antibodies were replaced with preimmune rabbit or mouse IgG.

Ida-Yonemochi H., Yamada Y., Yoshikawa H, & Seo K. (2017). Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury. PLoS ONE, 12(1), e0169201.

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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).

Hwang L., Ko I.G., Jin J.J., Kim S.H., Kim C.J., Chang B., Rho J.H., Moon E.J, & Yi J.W. (2019). Dexmedetomidine ameliorates memory impairment in sleep-deprived mice. Animal Cells and Systems, 23(6), 371-379.

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Western Blot Analysis of BDNF and TrkB

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Expression of BDNF and TrkB was determined by western blot analysis, as described previously [6 (link),20 (link)]. Hippocampal tissues were homogenized in lysis buffer. Protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto a nitrocellulose membrane. Rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology), and mouse anti-β-actin (1:1,000; Santa Cruz Biotechnology) antibodies were used as primary antibodies. Horseradish peroxidase-conjugated antimouse antibody for β-actin and antirabbit antibody for BDNF and TrkB were used as secondary antibodies.

Lee S.J., Kim T.W., Park H.K., Yoon S., You A.H., Moon E.J., Shin D.H, & Cho H. (2016). Postnatal Treadmill Exercise Alleviates Prenatal Stress-Induced Anxiety in Offspring Rats by Enhancing Cell Proliferation Through 5-Hydroxytryptamine 1A Receptor Activation. International Neurourology Journal, 20(Suppl 1), S57-64.

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Embryonic Tissue Immunolabeling Protocol

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E10.5 embryos were prepared for cryomicrotmy and antibody labeling as described previously (30 (link)). Primary antibodies include mouse anti-βIII Tubulin (TuJ1; Covance, 1:1000), rabbit anti-Six1 (Proteintech, 1:1500), chicken anti-GFP (Abcam1:1000), mouse anti-NeuN (Merck Millipore, 1:1000), mouse anti-HuC/D (16A11, Life Technologies, 1:1000), Mouse anti-BrdU (BD Biosciences, 1:100),goat anti-Sox10 (Santa Cruz Biotechnology, 1:50), mouse anti-Foxd3 (Thermo Scientific, 1:400), rabbit anti-Six4 (Proteintech Group, 1:100), mouse anti-Six1 (Atlas Antibodies, 1:200), mouse anti-Brn3a (Millipore, 1:100), mouse anti-p75 (Chemicon, 1:1000), rabbit anti-TrkA (Santa Cruz Biotechnology, 1:100), rabbit anti-TrkB (Santa Cruz Biotechnology, 1:100) and rabbit anti-cleaved Caspase 3 (Cell Signalling, 1:200). Primary antibody labeling was visualized with Alexafluor 488-, 546-, or 647- conjugated secondary antibodies (Molecular Probes, 1:2000 for 546 and 647, and 1:4000 for 488). Images were collected using a Leica Tiling microscope, or a Zeiss 710 confocal microscope.

Karpinski B.A., Bryan C., Paronett E., Baker J., Fernandez A., Horvath A., Maynard T.M., Moody S.A, & LaMantia A.S. (2016). A Cellular and Molecular Mosaic Establishes Growth and Differentiation States for Cranial Sensory Neurons. Developmental biology, 415(2), 228-241.

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Quantifying Hippocampal Protein Levels

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Western blot analysis for BDNF, TrkB, and AMPK was done as previously explained method [14 ,22 (link)]. Lysis buffer was applied to lyse hippocampal tissues, and protein content was detected by a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel was used to separated 30-μg protein, then reaction mixture was transferred to a nitrocellulose membrane, stopped reaction by applying dehydrated milk, and then incubated by primary antibodies. Mouse anti-β actin (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology), and rabbit anti-AMPK (1:1,000; Santa Cruz Biotechnology) were selected as the primary antibodies. Horseradish peroxidaseconjugated secondary antibodies were used, and enhanced chemiluminescence detection system (Santa Cruz Biotechnology) measured the expression of bands.

Kim S.H., Hwang L., Jin J.J., Ko I.G., Kim Y.B., Yoon H.S, & Baek S.S. (2021). Resistance Exercise Improves Spatial Learning Ability Through Phosphorylation of 5’-Adenosine Monophosphate-Activated Protein Kinase in Parkinson Disease Mice. International Neurourology Journal, 25(Suppl 2), S55-S62.

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Hippocampal Protein Expression Analysis

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Mice were killed by cervical dislocation at 16 weeks of age and their brains were collected and stored at −80°C. The hippocampus was bilaterally dissected and separated into dorsal and ventral hippocampus (~50/50). Protein extraction and Western blot analysis were performed as previously described (Hill et al., 2014 (link)). Primary antibodies were rabbit anti-BDNF (1:200, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA or Almone Labs, Israel), rabbit anti-NT-4 (1:200, Santa Cruz), rabbit anti-pTrkB Y705 (1:1,000, Signalway Antibody LLC, Maryland, USA), rabbit anti-pTrkB Y515 (1:1,000, Abcam, Cambridge, MA, USA), rabbit anti-pTrkB Y816 (1:500, Millipore, CA, USA), rabbit anti-TrkB (1:1,000, Santa Cruz), rabbit anti-NMDAR subunit 1 (GluNR1, 1:1,000, Cell Signaling Technology Inc, Danvers, MA, USA), rabbit NMDAR subunit 2A (GluN2A, 1:1,000, Cell Signaling Technology), rabbit NMDAR subunit 2B (GluN2B, 1:1,000, Cell Signaling Technology), or mouse anti-β-actin (1:10,000, Sigma-Aldrich). Secondary antibodies included anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies (1: 2,000; Cell Signaling Technology; Danvers, MA, USA).

Grech A.M., Ratnayake U., Hannan A.J., van den Buuse M, & Hill R.A. (2018). Sex-Dependent Effects of Environmental Enrichment on Spatial Memory and Brain-Derived Neurotrophic Factor (BDNF) Signaling in a Developmental “Two-Hit” Mouse Model Combining BDNF Haploinsufficiency and Chronic Glucocorticoid Stimulation. Frontiers in Behavioral Neuroscience, 12, 227.

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Immunostaining of Peripheral Nerve Tissues

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Nerves were explanted and de-sheeted, then fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich), included in OCT (Sakura, Leiden, The Netherlands) and cut in cross sections. For teased fibers, a slight digestion was performed incubating nerves fragments in collagenase IV for 45 min, before fixing in 4% PFA (Sigma-Aldrich). Cells were fixed in 4% PFA and processed for immunostaining. Primary antibodies used in these experiments were the following: rabbit anti PKCε 1:200 (Abcam), rabbit anti phospho S729 PKCε 1:200 (Abcam), mouse anti SMI31 1:500 (Biolegend, San Diego, CA, USA), mouse anti SMI32 1:500 (Biolegend), rabbit anti trkB 1:200 (Santa Cruz Biotechnology, Dallas, TX, USA) and fluoromyelin 1:150 (Thermo Fisher Scientific). After washing, slides and nerves were mounted using Vectashield^TM (Vector Laboratories, Burlingame, CA, USA) and nuclei stained with 4,6-diamidino-2-phenylindole (DAPI). Confocal microscopy was carried out using a Zeiss LSM 900 Airyscan 2 (Zeiss, Gottingen, Germany) and images were processed with Image Pro-Plus 6.0 (Media Cybernetics, Bethesda, MA, USA). Controls for specificity included a lack of primary antibody.

Bonalume V., Caffino L., Castelnovo L.F., Faroni A., Giavarini F., Liu S., Caruso D., Schmelz M., Fumagalli F., Carr R.W, & Magnaghi V. (2020). Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons. Cells, 9(8), 1874.

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Western Blot Analysis of BDNF Signaling

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20 μg of protein were separated by SDS-PAGE as described [8 ]. Mature BDNF (1:200 rabbit anti-BDNF; Santa Cruz), TRKB (1:500 rabbit anti-TrkB; Santa Cruz), phosphorylated TRKB at Y515 and Y817 (1:400 rabbit anti-TrkB phospho Y515; 1:1000 rabbit anti-TrkB phospho Y817; Abcam), p38 MAPK (1:1000 rabbit anti-p38; Cell Signaling), p42/44 MAPK (1:1000 rabbit anti-p42/44 (ERK1/2); Cell Signaling), phosphorylated p38 MAPK (1:1000 rabbit anti-Pp38; Cell Signaling), phosphorylated p42/44 MAPK (1:1000 rabbit anti-Pp42/44 (ERK1/2; Cell Signaling), and β actin (loading control; 1:1000 mouse anti-β-actin; Sigma) were measured by Western blotting. The membranes were then washed, incubated with the appropriate peroxidase conjugated secondary antibody, and visualized.

Prince C.S., Maloyan A, & Myatt L. (2016). Maternal obesity alters brain derived neurotrophic factor (BDNF) signaling in the placenta in a sexually dimorphic manner. Placenta, 49, 55-63.

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Hippocampal BDNF and TrkB Expression Analysis

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In the same manner, as described below, western blotting for the determination of BDNF and TrkB was conducted (Park et al., 2019 (link)). Hippocampal tissues were homogenized on ice and lysed in a lysis buffer consisting of 1-mM phenylmethylsulfonyl fluoride, 150-mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1% Nonidet P40, 100-mg/mL leupeptin, 50-mM Tris-HCl (pH, 7.5). Colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure protein content. Total protein 30 μg was separated on SDS-poly-acrylamide gels and transferred to a nitrocellulose membrane, the membrane was blocked with dehydrated milk and then treated with primary antibodies. Mouse anti-β actin (1:3,000; Santa Cruz Biotechnology, CA, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), and rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology) were used as the primary antibody.
After washing, appropriate horseradish peroxidase-conjugated secondary antibodies were used and incubation was performed at room temperature. Enhanced chemiluminescence detection system (Santa Cruz Biotechnology) was used to measure the expression of bands, and bands were quantified using an Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA).

Ji E.S., Kim Y.M., Ko Y.J, & Baek S.S. (2020). Treadmill exercise in obese maternal rats during pregnancy improves short-term memory through neurogenesis in the hippocampus of rat pups. Journal of Exercise Rehabilitation, 16(5), 392-397.

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Confocal Microscopy of Neuronal Proteins

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Neurons or SH-SY5Y cells were grown on poly-D-lysine treated glass coverslips and subjected to various treatments (e.g. differentiation, BDNF treatment). After treatment, cells were fixed in 4% paraformaldehyde in PBS, lysed with 0.3% Triton X-100 (5 minutes) and blocked in 10% donkey serum in Tris-buffered saline (pH 8.0) with 0.05% Tween 20 (TBST) overnight at 4°C. Fixed cells were then incubated overnight with antibodies (diluted in TBST) directed against various protein epitopes.
The following primary antibodies were used: mouse anti-Fyn (1:100; #15; Santa Cruz Biotechnology, Santa Cruz, California), rabbit anti-TrkB (1:100; #794; Santa Cruz Biotechnology), and mouse anti-FLAG (1:100; M2 monoclonal antibody, Stratagene, La Jolla, California). Coverslips were washed with TBST and the primary antibodies revealed by incubation with Alexa647-conjugated anti-mouse antibody (1:100) and Alexa555-conjugated anti-rabbit antibody (1:100; Invitrogen – Life Technologies). Images were captured using the Zeiss LSM 510 confocal microscope, using conditions to optimize the signal for each antibody used.
Confocal images were analyzed using ImageJ (NIH freeware) and the plug-in for colocalization analyses (Costes et al., 2004 (link)). The colocalization highlighter program is part of the same package and allows demarcation of colocalized regions under specified conditions.

Kao H.T., Ryoo K., Lin A., Janoschka S.R., Augustine G.J, & Porton B. (2017). Synapsins regulate BDNF-mediated synaptic potentiation and axon elongation by acting on membrane rafts. The European journal of neuroscience, 45(8), 1085-1101.

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