All lanes : Anti-MTCO1 antibody [1D6E1A8] (ab14705) at 0.5 µg/ml

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine heart at 1 µg
Lane 3 : Isolated mitochondria from Rat heart at 10 µg
Lane 4 : Isolated mitochondria from Mouse heart at 5 µg

Secondary
All lanes : Goat Anti-Mouse IgG

Predicted band size: 57 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?
Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.



Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.

IHC image of MTCO1 staining in human normal colon 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 ab14705, 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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Overlay histogram showing HEK293 cells stained with ab14705 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab14705, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

250 μm human cerebellar sections from control individuals and a patient with mitochondrial disease underwent passive clearing at 37 °C for 2 or 4 weeks.

The quality of immunofluorescent staining is determined by duration of passive clearing; 2 weeks of passive clearing produced minimal labelling of the white matter in the granule cell layer (NF-H; green; 488 nm and MBP; red, 546 nm) with an absence of labelling of mitochondria (MTCO1 (COXI) (ab14705, 1/100); purple; 647 nm; Extending passive clearing to 4 weeks improved the quality of stain with identifiable Purkinje cells and their axons (NF-H, green; 488 nm) and their myelin sheaths (MBP; red, 546 nm) and mitochondria (MTCO1 (COXI) (ab14705, 1/100); purple; 647 nm.