Anti-Cytochrome C antibody [7H8.2C12]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

ab13575 staining Cytochrome C in HepG2 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab13575 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

IHC image of Cytochrome C staining in human normal liver formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab13575, 1µg/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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

ab13575 staining human normal skin tissue. Staining is localised to mitochondria.
Left panel : with primary antibody at 4 ug/ml. Right panel : isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes 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 we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

Overlay histogram showing HepG2 cells stained with ab13575 (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 (ab13575, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1]/mouse IgG2b [PLPV219] (ab91353/ab91366, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• WB

Project

Western blot - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

Abcam recommends using milk (5%) as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes:

Western blot - Anti-Cytochrome C antibody [7H8.2C12] (ab13575) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

Human heart tissue lysate - total protein (<a href='/en-us/products/unavailable/human-heart-tissue-lysate-total-protein-ab29431'>ab29431</a>) at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 11 kDa

Observed band size: 14 kDa

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Exposure time: 3min

• WB

CiteAb

Western blot - Anti-Cytochrome C antibody [7H8.2C12] (AB13575)

Western Blotting using Anti-Cytochrome C antibody [7H8.2C12], ab13575. Publication image from Jia, Z. et al., 2019, Nucleic Acids Res, 30541130. Legend direct from paper.

Analysis of apoptosis. (A) The distributions of cytochrome c from control cybrid cell line (H25-03), mutant cybrid cell line (IV-3-13) and HUVECs were visualized by immunofluorescent labeling with cytochrome c antibody conjugated to Alex Fluor 488 (green) analyzed by a confocal fluorescence microscope. Mitotracker Red-stained mitochondria and DAPI-stained nuclei were identified by red and blue fluorescence respectively. (B) Twenty micorgrams of total proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with cytochrome c antibody with β-actin as a loading control. (C) Quantification of cytochrome c levels. The levels of cytochrome c in control and mutant cell lines were determined as described elsewhere (53). The calculations were based on three independent determinations in each cell line. (D) Twenty micrograms of total proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with cleaved PARP, Caspases 3, 7 and 9 antibodies with β-actin as a loading control. (E) Quantification of four apoptosis-activated proteins. The levels of Caspases 9, 3, 7 and PARP in various cell lines were determined as described elsewhere (52). The calculations were based on three independent determinations in each cell line. Graph details and symbols are explained in the legend to Figure 3.

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