ab24560 stained in SKNSH cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab24560 at 1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

All lanes : Anti-Hippocalcin antibody (ab24560) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Rat) Tissue Lysate
Lane 3 : Brain (Mouse) Tissue Lysate
Lane 4 : Mouse brain tissue lysate - total protein (0 days) (ab7188)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 23 kDa
Additional bands at: 14 kDa (possible cleavage fragment), 42 kDa, 52 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

Immuofluorescent staining for Hippocalcin in the rat hippocampus using Rabbit polyclonal to Hippocalcin (ab24560). This figure is a montage of pictures acquired with a X10 objective and shows expected abundance of staining in parts of the hippocampus such as the CA1 and CA3 and an absence of staining (as expected) in the cortex and the corpus callosum. ab24560 was used at 1/1000 (0.2µg/ml) incubated overnight at room temperature. Secondary antibody used was anti-rabbit Alexa Fluor 488 at 1/1000 incubated for 2 hours at room temperature. Rat brain tissue was perfusion fixed with 4% PFA followed by overnight post-fixation in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut on a cyrostat and immunohistochemistry performed by the 'free floating' technique.

Hippocalcin was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to Hippocalcin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab24560.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 23kDa: Hippocalcin; non specific - 60kDa: We are unsure as to the identity of this extra band.

All lanes : Anti-Hippocalcin antibody (ab24560) at 1 µg/ml

Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate (20ug)
Lane 3 : Mouse brain homogenate (20ug)

Secondary
All lanes : Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 23 kDa


Exposure time: 1 minute