Anti-Hsp60 antibody [1D11BD8] - Mitochondrial Marker (ab110312) at 5 µg/ml + HepG2 whole cells at 10 µg

Predicted band size: 61 kDa

ab110312 staining Hsp60 in MDA MB 231 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 10% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/100 in BSA + 0.02% Tween20) for 1 hour at 16°C. A DyLight^® 550-conjugated goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody.

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Immunocytochemistry image of stained fibroblast cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with the ab110312 antibody (1 µg/mL) for 2 hours at room temperature or over night at 4°C. The secondary antibody was (red) Alexa Fluor® 594 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent.

HeLa cells were stained with 1 µg/mL ab110312 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.

IHC image of Hsp60 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 ab110312, 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