Immunostained human cells in spleen and temporal artery (TA) xenograft sections demonstrate persistent human PBMC colonization at 28 days

Mitochondria in spleen (B) and temporal artery xenografts (E) were detected using ab92824 at 1/400 dilution in immunohistochemical analysis.

Magnification: 400X for spleen, 200X for TA grafts and 800X for insets.

(From Figure 2 of Chen et al)

Anti-Mitochondria antibody [113-1] (ab92824) at 1/1000 dilution + HeLa lysate

Observed band size: 60 kDa why is the actual band size different from the predicted?

ICC/IF image of ab92824 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab92824, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h.Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

IHC image of ab92824 staining in Breast Cancer 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 ab92824, 1/1000 dilution, 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 HepG2 cells stained with ab92824 (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 (ab92824, 1/100 dilution) 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 IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab92824 staining mitochondria in the Human U87 glioblastoma cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 10% NBF, permeabilized with Triton X-100 and blocked with 2% serum for 90 minutes at 22°C. Samples were incubated with primary antibody (1/1000 in 0.2% BSA + 2% NGS) for 15 hours at 4°C. An Alexa Fluor® 568-conjugated Goat anti-mouse IgG1 polyclonal was used as the secondary antibody (1/500). Co stained with ActinGreen, 2 drops/ml PBS, 10 min and Hoechst 2ug/ml in H2O 10 min

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