Immunocytochemistry/ Immunofluorescence - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

ab14705 staining MTCO1 - Mitochondrial Marker in pig retinal pigment epithelial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% TX-100 and blocked with 5% serum for 20 minutes at 25ºC. Samples were incubated with primary antibody (1/2000 in 1% goat serum, 0.1% TX100; PBS) for 16 hours at 4ºC. An Alexa Fluor^®546-conjugated Goat polyclonal to mouse IgG, dilution 1/500, was used as secondary antibody.

This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic

Immunocytochemistry/ Immunofluorescence - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

ab14705 staining MTCO1 - Mitochondrial Marker in rat cerebellum primary cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% TX-100 and blocked with 5% serum for 20 minutes at 25ºC. Samples were incubated with primary antibody (1/2000 in 1% goat serum, 0.1% TX100; PBS) for 16 hours at 4ºC. An Alexa Fluor^®546-conjugated Goat polyclonal to mouse IgG, dilution 1/5000, was used as secondary antibody.

This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic

Flow Cytometry - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

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.

Western blot - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

All lanes : Anti-MTCO1 antibody [1D6E1A8] - Mitochondrial Marker (ab14705)

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
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.

Immunohistochemistry (Frozen sections) - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

ab14705 staining MTCO1 in skeletal muscle tissue by Immunohistochemistry (Frozen sections). Tissue sections were from from a patient with a single large deletion of the mtDNA and show a mosaic of complex IV positive and complex IV negative fibers.

Immunohistochemistry (Frozen sections) - MTCO1 antibody [1D6] - Mitochondrial Marker (ab14705)

ab14705 staining MTCO1 in Human colon tissue by Immunohistochemistry (Frozen sections). Tissue sections from a normal ageing patient show complex IV negative crypts due to clonal expansion of colonic stem cells bearing mutations in the mtDNA-encoded gene for complex IV.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTCO1 antibody [1D6E1A8] - Mitochondrial Marker (ab14705)

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTCO1 antibody [1D6E1A8] - Mitochondrial Marker (ab14705)

ab14705 staining rat pancreas sections by IHC-P. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1% BSA for 10 minutes at 21°C, followed by staining with ab14705 at 1/1000 in TBS/BSA/azide for 2h at 21°C. A biotinylated goat anti-mouse polyclonal antibody at 1/200 was used as the secondary antibody. Positivity in exocrine glands appears to be intense at the cytplasm of adjacent cells. The cells of the Islet of Langerhan to the right have a diffuse, punctate positivity.

This image is courtesy of an Abreview submitted by Carl Hobbs