Dopamine Receptor D2
| Cat Number: | AB-11173 |
|---|---|
| Conjugate: | Unconjugated |
| Size: | 100 ul |
| Clone: | POLY |
| Concentration: | 1mg/ml |
| Host: | Rb |
| Isotype: | IgG |
| Reactivity: | Hu, Rt, Ms |
| Applications: | Elisa, Western Blot, Immunocytochemistry, Immunohistochemistry (frozen tissues) |
| Purification: | Serum |
| Background: | The antiserum was raised in a rabbit which was immunized with D2 (272-282) covalently attached onto a carrier protein, and it has been characterized by immunocytochemical, western immunoblot and ELISA techniques. The antiserum has been found to be highly specific for this peptide sequence and is suitable for immunocytochemical detection of both the short and the long forms of the D2 dopamine receptor. Rehydrate the antibody with 0.1 ml of PBS which contains 10 mg/ml BSA or with additional buffer (such as 10 mg/ml BSA in PBS) for more dilute antibody.100% cross reactivity with D2 Dopamine Receptor (272-282), ~ 70% with D2S Dopamine Receptor, ~ 60% with D2L No cross reactivity with the other Dopamine Receptors. |
| Form: | Liquid |
Working Dilutions Primary Antibody: 1:300
Tests performed by Fusco F.R., Giampà C. Neuroanatomy Lab. Fondazione S. Lucia, Roma
Working Dilutions Primary Antibody: 1:300
Tests performed by Fusco F.R., Giampà C. Neuroanatomy Lab. Fondazione S. Lucia, Roma
Protocol followed by Fusco F.R., Giampà C. Neuroanatomy Lab. Fondazione S. Lucia, Roma
Tissue processing
The animals (rats or mice) are transcardially perfused under deep anaesthesia with chloral hydrate with 60 mL of saline solution containing 0.05 mL heparin, followed by 200 mL of 4% paraformaldehyde in saline solution.
The brains are removed and postfixed overnight at +4 °C, cryoprotected in 10% sucrose and 20% glycerol in 0.1 m phosphate buffer (PB) with sodium azide 0.02% for 48 h at 4 °C. Brains were sectioned frozen on a sliding microtome at 40 lm thickness.
Single label Immunofluorescence
• Sections are rinsed for 15 min in PB containing TritonX-100 0.3%
• Sections, after a pre-incubation with the appropriate normal serum, are incubated overnight with a primary antibody at the dilution tested (1:200-1:500) in 0.1 M PB containing Triton X-100 0.3%, azide 0.02% at 4° C for three night.
• Subsequently, sections are rinsed three times for 15 min in 0.1 M PB and then incubated with Alexa Fluor 488-conjugated secondary antibody for 2 h at room temperature.
• Sections are rinsed three times for 15 min in 0.1 M PB.
• Tissue are mounted on gelatin-coated slides, coverslipped with GEL-MOUNT™ and examined under an epiillumination fluorescence.
Western Blotting Protocol
1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels, such at 12% gels) and electrophoretic transfer to PVDF membrane, block the membrane overnight with 2% normal goat serum in TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the rabbit polyclonal antibody after dilution to at least 1:800 (Note: higher dilutions may be needed). Use 2% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. Let the primary antibody bind for 2-4 hours.
4. Wash x 3 with TBS/Tween-20.
5. Apply affinity purified HRP-goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash x 4 for 5 minute/wash with TBS/Tween-20.
7. Develop color using the enhanced DAB reaction.
Immunocytochemistry Protocol, on cells