Confocal image showing nuclear staining on F9 cells

Ab92494 staining SOX2 in the F9 (mouse embryonal carcinoma) cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X. Samples were incubated with primary antibody (1/200). An Alexa Fluor^® 488-conjugated Goat anti-Rabbit IgG, Ab150077 (1/1000) was used as the secondary antibody. Counterstained with Ab7291 anti-Tubulin (1/1000), Ab150120 AlexaFluor^®594 Goat anti-Mouse secondary (1/1000). DAPI was used as a nuclear counter stain.

Negative control 1 Ab92494 was used as the primary antibody at 1/200 and Ab150120 was used as the secondary at 1/1000.

Negative control 2 Ab7291was used as the primary antibody at 1/1000 and Ab150077 was used as the secondary at 1/1000.

All lanes : Anti-Nestin antibody [Rat 401] (ab6142) at 1 µg/ml

Lane 1 : Mouse E12 brain tissue lysate

Lane 2 : Mouse E14 brain tissue lysate

Lane 3 : Mouse E16 brain tissue lysate

Lane 4 : Mouse E18 brain tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 200 kDa

This blot was produced using a 3-8% Tris-Acetate gel under the Tris-Acetate 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 3% milk before being incubated with ab6142 overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-Notch1 antibody [EP1238Y] (ab52627) at 1/2000 dilution (Purified) + Mouse brain at 10 µg

Secondary

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Observed band size: 125 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on distal tubules of mouse kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

ab11512 stained in M158 cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab11512 at 10 µg/ml overnight at +4°C. The secondary antibody was ab150165 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

Sample: Murine derived breast cancer whole cell lysate, 30 µg.

Anti-E Cadherin antibody [DECMA-1] (ab11512) used at a 1/5000 dilution for 16 hours at 4°C.

Goat polyclonal IRDye 800CW used as the secondary antibody at a 1/10,000 dilution.