IHC image of Prohibitin staining in human liver FFPE section, performed on a 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 ab28172, 5µg/ml, for 8 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.

All lanes : Anti-Prohibitin antibody - Mitochondrial Marker (ab28172) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Brain (Mouse) Tissue Lysate
Lane 4 : Liver (Mouse) Tissue Lysate - normal tissue
Lane 5 : Heart (Mouse) Tissue Lysate
Lane 6 : Kidney (Mouse) Tissue Lysate
Lane 7 : Mouse pancreas tissue lysate - total protein (ab29363)
Lane 8 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 9 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
Lane 10 : Spinal Cord (Mouse) Tissue Lysate
Lane 11 : Ovary (Mouse) Tissue Lysate - normal tissue
Lane 12 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 13 : Brain (Rat) Tissue Lysate - normal tissue
Lane 14 : Liver (Rat) Tissue Lysate
Lane 15 : Heart (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 30 kDa

ab28172 staining Prohibitin in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Triton for 1h. The cells were then incubated with the antibody ab28172 at 5µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

ab28172 staining Prohibitin in mouse liver tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in Citrate buffer pH 6.0 and blocking in 1.5% serum for 10 minutes. The primary antibody was diluted, 1/400 and incubated with sample for 1 hour. A HRP conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

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All lanes : Anti-Prohibitin antibody - Mitochondrial Marker (ab28172) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat whole cell lysate (ab7899)
Lane 3 : A-431 whole cell lysate (ab7909)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 30 kDa

ICC/IF image of ab28172 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab28172, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used to quench autofluorescence.  5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).