ab216880 stained in MCF7 (Human breast adenocarcinoma cell line) cells. Cells were fixed with 100% methanol (5 minutes) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab216880 at 1 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (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 1 hour at room temperature

All lanes : Anti-ZO1 tight junction protein antibody (ab216880) at 1 µg/ml

Lanes 1 & 9 : Protein Standard at 10 µl
Lane 2 : WM115 (human melanoma cell line) whole cell lysate at 10 µg
Lane 3 : A549 (human lung carcinoma cell line) whole cell lysate at 10 µg
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Lane 5 : PC-3 (human prostate adenocarcinoma cell line) whole cell lysate at 10 µg
Lane 6 : SK-OV-3 (human ovarian cancer cell line) whole cell lysate at 10 µg
Lane 7 : Mouse testis tissue lysate at 10 µg
Lane 8 : Rat testis tissue lysate at 10 µg

Secondary
All lanes : Goat anti Rabbit IgG H&L (HRP) at 1/70000 dilution

Performed under reducing conditions.

Predicted band size: 195 kDa
Observed band size: 187,195 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Lysates reduced with beta-Mercaptoethanol and proteins separated by SDS-PAGE. Transferred to 0.2 µm nitrocellulose membrane at 20V for 7 minutes. Blocked with 5% BLOTTO for 1 hour at room temperature. Incubated with primary overnight at 2-8°C, followed by secondary for 30 minutes at room temperature.

Immunocytochemistry analysis of 4% paraformaldehyde fixed Caco-2 (human colorectal adenocarcinoma cell line) cells labeling ZO1 tight junction protein with ab216880 at 15 µg/mL. Cells permeabilized with 0.3% Triton X-100 and incubated with primary antibody overnight at 2-8°C. Donkey Anti-Rabbit IgG DyLight™ 488 Conjugated (Preadsorbed) was used as the secondary antibody at 5 µg/mL for 1 hour at room temperature. Staining: (A) DAPI. (B) ab216880 with secondary. (C) Merge A-B. (D) Secondary Only.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded mouse adipose tissue labeling ZO1 tight junction protein with ab216880 at 1/100 dilution (A) or 1/200 dilution (B). Visualized with WARP RED on MACH 4 universal AP polymer detection system.

Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections) analysis of mouse adipose tissue and muscle labelling ZO1 tight junction protein with ab216880 at 1/100 dilution. Antigen retrieval: heat induced (HIER) using Citrate Buffer for 20min. Primary antibody incubation for 30 minutes at room temperature followed by incubation with Anti-Rabbit IgG (HRP) secondary antibody for 8 minutes at room temperature. Localization: ZO-1 will stain cell-cell junctions. Staining: DAB. Counter Stain: Hematoxylin.

Image A: H&E staining (no primary)

Image B: Primary antibody present. 20X magnification

Image C: Primary antibody present. 40X magnification

 

Immunofluorescent analysis of 0.5% PFA-fixed (A and C) and 0.5% methanol-fixed (B and D) Caco-2 (human colorectal adenocarcinoma cell line) cells labeling ZO1 tight junction protein with ab216880 at 10 µg/ml (green).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control: C and D.