ab14613 labels all PDE4D proteins including PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5 variants. Does not cross react with PDE4A, PDE4B, PDE4D6, PDE4D7, PDE4D8, PDE4D9 or PDE4C.
The antibody has not been tested against endogenous PDE4D, only recombinant proteins and we therefore cannot guarantee it will detect endogenous PDE4.
ab14613 (2µg/ml) staining PDE4D in human placenta using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of the cytoplasm. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
ICC/IF image of ab14613 stained HeLa cells. The cells were 4% formaldehyde 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 (ab14613, 10µg/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.
Western blot - Anti-PDE4D antibody (ab14613)
Western blot using ab14613 and positive controls for PDE4D variants.
Cheng H et al. siRNA-mediated silencing of phosphodiesterase 4B expression affects the production of cytokines in endotoxin-stimulated primary cultured microglia. Exp Ther Med12:2257-2264 (2016).
Read more (PubMed: 27698721) »
Barbagallo F et al. Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure. Circ Res119:931-43 (2016).
Read more (PubMed: 27576469) »