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

5 product images

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  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 Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) 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.

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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) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1]/mouse IgG2b [PLPV219] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353/Mouse IgG2b [PLPV219] - Isotype Control 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.

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  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 (ab29431) at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040) at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 11 kDa

  Observed band size: 14 kDa

  Exposure time: 3min

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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.

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  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.