Anti-NOTCH3 antibody

• WB

Ap10489****

Western blot - Anti-NOTCH3 antibody (AB23426)

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab23426 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

The band observed at 97 kDa is thought to correspond to the notch-derived peptide containing the intracellular domain (NICD) of NOTCH3 as described in the literature (PMID : 10712431).

All lanes:

Western blot - Anti-NOTCH3 antibody (ab23426) at 1 µg/mL

Lane 1:

K-562 whole cell lysate (<a href='/en-us/products/unavailable/k-562-whole-cell-lysate-ab29306'>ab29306</a>) at 10 µg

Lane 2:

Caco-2 whole cell lysate (<a href='/en-us/products/unavailable/caco-2-whole-cell-lysate-ab3950'>ab3950</a>) at 10 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L (HRP) at 1/50000 dilution

Predicted band size: 244 kDa

Observed band size: 105 kDa,280 kDa,30 kDa,97 kDa

true

Exposure time: 30s

• IHC-P

AbReview12463****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody (AB23426)

ab23426 staining NOTCH3 in human breast cancer tissue sections by immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue underwent fixation in paraformaldehyde, heat-mediated antigen retrieval in citrate buffer pH6.0 and blocking for 15 minutes at 20°C (5 minutes for peroxidase blocking and 10 minutes for protein blocks). The primary antibody was diluted 1/250 and incubated with sample for 45 minutes at 20°C. A HRP-conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody (AB23426)

IHC image of NOTCH3 staining in human breast carcinoma 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 ab23426, 5µg/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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NOTCH3 antibody (AB23426)

ICC/IF image of ab23426 stained MCF7 cells. The cells were 100% methanol fixed (5 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 (ab23426, 5μg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight^® 488 goat anti-rabbit 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-P

AbReview80633****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NOTCH3 antibody (AB23426)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of paraformaldehyde fixed human tonsil permeabilized with 0.3% Triton X-100, staining with ab279690 at 2µg/ml. Secondary antibody used was ab 150075 at 1/500 dilution. Blocking was done using Donkey Serum 10% + 3% BSA for 24 hours at 4°C. The sample was incubated with the primary antibody for 1 hour at 20°C. Validated on GE Cell DIVE. Antigen retrieval method was heat mediated Citrate pH 6 & TRIS pH 9

This image is courtesy of an Abreview submitted by Dylan Windell

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NOTCH3 antibody (AB23426)

ab23426 staining NOTCH3 in mES cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab23426 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

AbReview34530****

Immunocytochemistry/ Immunofluorescence - Anti-NOTCH3 antibody (AB23426)

ab23426 staining NOTCH3 in the COS1 fibroblast cell line from Monkey Kidney by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% in PBS and blocked with 1% BSA for 30 minutes at 25°C. Samples were incubated with primary antibody (1/200) for 16 hour at 4°C. An Alexa Fluor^® 488-conjugated Goat anti-rabbit polyclonal (1/500) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview