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TrkB (80G2) Rabbit mAb #4607
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using TrkB (80G2) Rabbit mAb.Learn more about how we get our images
Gallery: TrkB (80G2) Rabbit mAb #4607
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%)
- Deionized water (dH2O)
- Hematoxylin (optional)
- Wash Buffer:
- 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
- SignalStain® Antibody Diluent (#8112).
- Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7â¢2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
- Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
- Substrate: Vector® NovaRED™ (Vector Laboratories).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 minutes each.
- Incubate sections in two washes of 100% ethanol for 10 minutes each.
- Incubate sections in two washes of 95% ethanol for 10 minutes each.
- Wash sections twice in dH2O for 5 minutes each.
C. Antigen Unmasking
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 minutes each.
- Incubate sections in 3% hydrogen peroxide for 10 minutes.
- Wash sections in dH2O twice for 5 minutes each.
- Wash sections in wash buffer for 5 minutes.
- Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
- Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Prepare ABC solution per manufacturer's recommendations.
- Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
- Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
- Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
- Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
- Wash section three times with wash buffer for 5 min each.
- Prepare Vector® NovaRED™ per manufacturer's recommendations.
- Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 minutes each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 seconds each.
- Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
- Repeat in xylene, incubating sections two times for 10 seconds each.
- Mount coverslips.
posted June 2005
revised February 2008
protocol id: 303
A. Solutions and Reagents
- 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
- Fix for 10 minutes at 37°C.
- Add 5 ml PBS and rinse by centrifugation.
- Resuspend cells in 5 ml PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies
NOTE: Allow species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.
- Aliquot 0.5-1x106 cells into each assay tube (by volume).
- Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
- Resuspend cells in 100 μl Incubation Buffer per assay tube.
- Block in Incubation Buffer for 10 minutes at room temperature.
- Add the primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
- Incubate for 30-60 minutes at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in fluorochrome-conjugated secondary antibody*, diluted in Incubation Buffer according to the manufacturer’s recommendations.
- Incubate for 30 minutes at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
*Recommended Secondary Antibodies:
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4413
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
posted January 2009
protocol id: 133
TrkB (80G2) Rabbit mAb detects endogenous levels of total TrkB protein. The antibody does not cross-react with TrkA.Species Reactivity: Human Species predicted to react based on 100% sequence homology: Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide surrounding Pro50 of human TrkB.
The family of Trk receptor tyrosine kinases consists of TrkA, TrkB, and TrkC. While the sequence of these family members is highly conserved, they are activated by different neurotrophins: TrkA by NGF, TrkB by BDNF or NT4, and TrkC by NT3 (1). Neurotrophin signaling through these receptors regulates a number of physiological processes, such as cell survival, proliferation, neural development, and axon and dendrite growth and patterning (1). In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. TrkA regulates proliferation and is important for development and maturation of the nervous system (2). Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAP kinase cascade (3,4). Residues Tyr674/675 lie within the catalytic domain, and phosphorylation at these sites reflects TrkA kinase activity (3-6). Point mutations, deletions, and chromosomal rearrangements (chimeras) cause ligand-independent receptor dimerization and activation of TrkA (7-10). TrkA is activated in many malignancies including breast, ovarian, prostate, and thyroid carcinomas (8-13). Research studies suggest that expression of TrkA in neuroblastomas may be a good prognostic marker as TrkA signals growth arrest and differentiation of cells originating from the neural crest (10).
The phosphorylation sites are conserved between TrkA and TrkB: Tyr490 of TrkA corresponds to Tyr512 in TrkB, and Tyr674/675 of TrkA to Tyr706/707 in TrkB of the human sequence (14). TrkB is overexpressed in tumors, such as neuroblastoma, prostate adenocarcinoma, and pancreatic ductal adenocarcinoma (15). Research studies have shown that in neuroblastomas, overexpression of TrkB correlates with an unfavorable disease outcome when autocrine loops signaling tumor survival are potentiated by additional overexpression of brain-derived neurotrophic factor (BDNF) (16-18). An alternatively spliced truncated TrkB isoform lacking the kinase domain is overexpressed in Wilms’ tumors and this isoform may act as a dominant-negative regulator of TrkB signaling (17).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 5,675,063.