Anti-CD133 antibody [RM1029]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labelling CD133 with ab284389 at 1/500 (1.016 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Apical staining on human breast. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Human pancreatic carcinoma tissue labelling CD133 with ab284389 at 1/500 (1.016 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Apical staining on human pancreatic carcinoma. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling CD133 with ab284389 at 1/500 (1.016 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on parietal layer of Bowman's capsule and apical staining on renal tubules in human kidney. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling CD133 with ab284389 at 1/500 (1.016 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on the pancreatic ducts in human pancreas. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD133 antibody [RM1029] (AB284389)

Flow cytometric analysis of 293T (Human embryonic kidney epithelial cell, Left), Caco-2 (Human colorectal adenocarcinoma epithelial cell, Right) cells labelling CD133 with ab284389 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/2000 dilution was used as the secondary antibody. Negative control : 293T. Gated on viable cells.

• ICC

Lab

Immunocytochemistry - Anti-CD133 antibody [RM1029] (AB284389)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Caco-2 cells labelling CD133 with ab284389 at 1/50 (10.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranous staining in Caco-2 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IP

Lab

Immunoprecipitation - Anti-CD133 antibody [RM1029] (AB284389)

CD133 was immunoprecipitated from 0.35 mg Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate 10ug with ab284389 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab284389 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab284389 IP in Caco-2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab284389 in Caco-2 whole cell lysate

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

Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-CD133 antibody [RM1029] (ab284389)

Predicted band size: 97 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labelling CD133 with ab284389 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on the biliary ducts of mouse liver. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse E14.5 embryonic kidney tissue labeling CD133 with ab284389 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution (Green). Positive staining on the apical side of mouse E14.5 embryonic kidney buds is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/ml dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Mouse breast tissue labelling CD133 with ab284389 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Apical staining on mouse breast. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD133 antibody [RM1029] (AB284389)

Flow cytometric analysis of Mouse bone marrow cell cells labelling CD133 with ab284389 at 1/50 dilution (1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab271092. Then stained with Lineage cocktail conjugated to FITC, anti-Sca-1 conjugated to PE/Cy7, anti-CD117 conjugated to APC/Cy7 and anti-CD16/32 conjugated to BV510.Gated on viable MP cells. Positive staining on CD16/32+ MP cells is consistent with literature. (PMID : 23509298)

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse E14.5 embryonic lung tissue labeling CD133 with ab284389 at 1/100 (5.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution (Green). Positive staining on the apical side of mouse E14.5 embryonic lung buds is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/ml dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD133 antibody [RM1029] (AB284389)

Immunohistochemical analysis of paraffin-embedded Mouse retina tissue labelling CD133 with ab284389 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on the cone and rod cells of mouse retina. The section was incubated with ab284389 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD133 antibody [RM1029] (AB284389)

Flow cytometric analysis of Mouse bone marrow cell cells labelling CD133 with ab284389 at 1/50 dilution (1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/2000 dilution was used as the secondary antibody. Cells were co-stained with anti-CD117 conjugated to APC/Cy7. Gated on viable cells.

• IP

Lab

Immunoprecipitation - Anti-CD133 antibody [RM1029] (AB284389)

CD133 was immunoprecipitated from 0.35 mg Mouse kidney lysate 10ug with ab284389 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab284389 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse kidney lysate 10ug

Lane 2 : ab284389 IP in Mouse kidney lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab284389 in Mouse kidney lysate

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

Exposure time : 5 seconds.

All lanes:

Immunoprecipitation - Anti-CD133 antibody [RM1029] (ab284389)

Predicted band size: 97 kDa

false

• WB

Lab

Western blot - Anti-CD133 antibody [RM1029] (AB284389)

Blocking and diluting buffer and concentration : 5%

NFDM/TBST Negative control : MEF (PMID : 21709693)

Similar band pattern was observed in literature. (PMID : 26137114)

Lysate were not boiled to avoid protein aggregation.

All lanes:

Western blot - Anti-CD133 antibody [RM1029] (ab284389) at 1/1000 dilution

Lane 1:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

ES-D3 (Mouse embryo spherical colony) whole cell lysate at 20 µg

Lane 5:

MEF (Mouse embryonic fibroblast (immortalized) whole cell lysate at 20 µg

Lane 6:

F9 (Mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

Mouse E12.5 brain lysate at 20 µg

Lane 8:

Rat E12.5 brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 97 kDa

false

Exposure time: 1min