The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 28 kDa (predicted molecular weight: 28 kDa).
Dynorphins are a class of peptides produced by many different populations of neurons, which has some opiate like activity. It is thus classed as an endogenous opioid peptide. The dynorphins, which include dynorphin A, dynorphin B, alpha and beta neoendorphin, and big dynorphin, are all the products of a single gene, 'preprodynorphin'. Dynorphin is produced in many different parts of the brain, including the hypothalamus, the hippocampus and the spinal cord, and has many different physiological actions, depending upon its site of production. Dynorphin is involved in modulating responses to several psychoactive substances, including cocaine. As such, it may help some individuals against addiction. Blocking dynorphin may help alleviate depression.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dynorphin A antibody (ab82509)
IHC image of Dynorphin A staining in formalin fixed, paraffin embedded mouse dorsal root ganglion tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab82509, 10µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Western blot - Dynorphin A antibody (ab82509)
Anti-Dynorphin A antibody (ab82509) at 1 µg/ml + Human brain normal tissue lysate - membrane extract (ab29456) at 10 µg
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 28 kDa Observed band size: 28 kDa
Exposure time: 15 minutes
Immunocytochemistry/ Immunofluorescence - Dynorphin A antibody (ab82509)
ICC/IF image of ab82509 stained PC12 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab82509, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) PC12 cells at 5µg/ml.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Dynorphin A antibody (ab82509)
IHC image of Dynorphin A staining in human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab82509, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX
Western blot - Anti-Dynorphin A antibody (ab82509)This image is courtesy of an anonymous Abreview
Anti-Dynorphin A antibody (ab82509) at 1/1000 dilution + Mouse synaptosome from hippocampal stratum lucidum tissue lysate at 10 µg
Secondary HRP-conjugated anti-rabbit IgG polyclonal at 1/5000 dilution