All lanes : Anti-HADHA antibody (ab54477) at 4 µg/ml

Lanes 1-2 : Jurkat cell lysate 30-50 ug/lane.
Lane 3 : 3T3 cell lysate 30-50 ug/lane.
Lane 4 : Rat kidney lysate 30-50 ug/lane.

Secondary
All lanes : Anti-Rabbit IgG, HRP-Linked Antibody at 1/5000 dilution

Predicted band size: 83 kDa
Observed band size: 83 kDa

ICC/IF image of ab54477 stained HepG2 cells. The cells were 100% methanol fixed (5 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 (ab54477, 1µg/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.

ab54477 (1µg/ml) staining HADHA in human ileum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong mitochondrial staining.
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.