ab23426 staining NOTCH3 in mouse embryo tissue (E16.5) by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue underwent fixation in formaldehyde, heat-mediated antigen retreival in citrate buffer pH6.0 and blocking for 15 minutes at 10°C (5 minutes/peroxidase blocking and 10 minutes/protein blocking). The primary antibody was diluted 1/250 and incubated with sample for 15 minutes at 20°C. A HRP-conjugated goat polyclonal to rabbit IgG was used undiluted as the secondary.See Abreview

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.See Abreview

IHC-FoFr image of NOTCH3 staining in Rat cerebral cortex (left) and the paraventricular hypothalamic nucleus (right) sections. The sections used came from animals perfused fixed with Paraformaldehyde 4%, in phosphate buffer 0.2M. Following postfixation in the same fixative overnight, the brains were cryoprotected in sucrose 30% overnight. Immunostainings were performed using the ‘free floating’ technique. See Abreview

IHC image of NOTCH3 staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 image of ab23426 stained mouse embryonic stem cells. The cells were 4% PFA fixed (10 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.

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.

ab14404 staining NOTCH3 in the COS1 fibroblast cell line from Monkey Kkidney 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.See Abreview