Recombinant Anti-CD133 antibody [RM1002] - Stem Cell Marker (ab278053)

IHC image of CD133 staining in a section of frozen normal human tonsil* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab278053, 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. The inset secondary-only control image is taken from an identical assay without primary antibody.
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab278053 was shown to react with PROM1 in wild-type HAP1 cells in western blot. Loss of signal was observed when PROM1 knockout sample was used. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab278053 overnight at 4°C at a 1 in 1000 dilution. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) secondary antibody at 1/5000 for 1 hour at room temperature before development with Optiblot ECL reagent (ab133456) and imaging.

Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling CD133 with ab278053 at 1/1000 (0.5 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Apical staining on human mammary gland (PMID: 18261235). The section was incubated with ab278053 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD133 with ab278053 at 1/1000 (0.5 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on Bowman’s capsule of human kidney (PMID: 19875807). The section was incubated with ab278053 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of HEK-293T (Human embryonic kidney epithelial cell, Left panel) / Caco-2 (Human colorectal adenocarcinoma epithelial cell, Right panel ) using ab278053 at 1/500 dilution (0.1µg) (Red). Mouse monoclonal IgG  (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti mouse IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: HEK-293T. (PMID 20167130)
Gated on viable cells.

Blocking/Dilution buffer: 5% NFDM/TBST.

Negative control: HEK-293T (PMID 20167130).

The MW observed is consistent with the literature (PMID 20717901; PMID 18645205).

CD133 was immunoprecipitated from 0.35 mg Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab278053 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278053 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Caco-2 whole cell lysate 10 μg.

Lane 2: ab278053 IP in Caco-2 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab278053 in Caco-2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds.