Anti-CaMKII alpha (phospho T286) antibody

• IHC-FoFr

Collaborator

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-CaMKII alpha (phospho T286) antibody (AB5683)

Immunofluorescent staining for CaMKII alpha phospho (T286) using ab5683 (1/300, incubated for 18 hours) in rat spinal cord. To induce CaMKII alpha phospho (T286) protein expression, a noxious stimulus was administered to the rat 5 minutes prior to 4% PFA perfusion fixation (this is a known paradigm for inducing phosphorylation CaMKII in some spinal neurons). The resulting immunofluorescent staining for CaMKII alpha phospho (T286) protein is observed in the cytoplasm of many dorsal horn spinal neurons (surrounding the central canal [A] or in the ventral horn [B]). Omition of primary antibody resulted in a lack of staining (data not shown).

Protocol details : Tissue was prepared by 4% paraformaldehyde cardiac perfusion fixation. Tissue was frozen on dry ice and then embedded in OCT compound and cut on cryostat. An antigen retrieval step was not neccesary for the IHC protocol.

This image is courtesy of Sophie Pezet, Univ London Kings Coll, United Kingdom

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CaMKII alpha (phospho T286) antibody (AB5683)

ICC/IF image of ab5683 stained PC12 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 (ab5683, 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.