Anti-NOTCH3 antibody [EPR25183-101]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

Immunohistochemical analysis of paraffin-embedded Panel A HEK-293T c tissue labeling NOTCH3 with ab300527 at 1/1000 (0.506 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on (A) HEK-293T transfected with a human NOTCH3 expression vector, no staining on (B) HEK-293T transfected with empty vector. The section was incubated with ab300527 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 (Intra)

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Flow Cytometry (Intracellular) - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MDA-MB-231 (human breast adenocarcinoma epithelial cell, Left) / MCF7 (human breast adenocarcinoma epithelial cell, Right) cells labelling NOTCH3 with ab300527 at 1/50 dilution (1ug) (Red) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Low expression : MDA-MB-231(PMID : 29109797).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

Immunohistochemical analysis of paraffin-embedded Human smooth muscle tissue labeling NOTCH3 with ab300527 at 1/500 (1.012 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on vascular smooth muscle cells in human smooth muscle. The section was incubated with ab300527 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling NOTCH3 with ab300527 at 1/500 (1.012 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human lung carcinoma. The section was incubated with ab300527 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling NOTCH3 with ab300527 at 1/500 (1.012 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on vascular smooth muscle cells of human enodmetrium. The section was incubated with ab300527 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

• WB

Lab

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (ab300527) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (frozen) at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (fresh) 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 1/20000 dilution

Observed band size: 90 kDa,270 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (ab300527) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) transfected with scrambled siRNA control, whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeti NOTCH3, whole cell 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

Observed band size: 90 kDa,270 kDa

true

Exposure time: 169s

• WB

Supplier Data

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (AB300527)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-NOTCH3 antibody [EPR25183-101] (ab300527) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lane 2:

MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate

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

Observed band size: 90 kDa,270 kDa

true

Exposure time: 48s