ab40772 staining E Cadherin in HT-29 (Human colorectal adenocarcinoma) cells by ICC/IF (Immunocytochemistry/Immunofluorescence).  Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were incubated with primary antibody at 1/500 dilution. An Alexa Fluor^® 488 Goat anti-Rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution was used as a counterstain. DAPI was used as a nuclear counterstain. This is a confocal image showing membranous staining on HT-29 cell line.

Confocal image showing nuclear staining on NCCIT cells

Ab92494 staining SOX2 in NCCIT cells 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-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate at 40 µg

Lane 2 : OCLN (Occludin) knockout HAP1 whole cell lysate at 40 µg

Lane 3 : HeLa whole cell lysate (Low Occludin expression) at 20 µg

Lane 4 : HepG2 whole cell lysate lysate (High Occludin expression) at 20 µg

Predicted band size: 59 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab216327 observed at 59 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab216327 was shown to recognize Occludin in wild-type HAP1 cells as signal was lost at the expected MW in OCLN (Occludin) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and OCLN (Occludin) knockout samples were subjected to SDS-PAGE. Ab216327 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at  1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Occludin expression in HeLa is expected to be negative.

Formalin/PFA-fixed paraffin-embedded human colonic adenocarcinoma tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Immunofluorescent staining of SH-SY5Y cells (fixed with 4% PFA and permeablized with TritonX 100) with purified ab108937 at a dilution of 1/100. An Alexa Fluor^® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.

Primary ab concentration dilution: 1:200, (0.5ug/ml), Secondary ab: ImmunoHistoprobe (Ready to use) HRP Polymer for Rabbit IgG, Secondary ab concentration: Prediluted, Tissue: Human brain, Fixative: Paraffin-embedded sections, Counter stain: Hematoxylin Antigen retrieval: Perform heat mediated antigen retrieval using Tris/EDTA Buffer, PH9

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cells labeling Occludin with ab216327 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membrane staining on Caco-2 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on human colon is observed (PMID: 24268521). 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.

Immunohistochemical staining of paraffin-embedded human brain with purified ab52627 at a dilution of 1/150.

A prediluted HRP polymer for rabbit IgG was used as the secondary and the sample was stained with hematoxylin. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells fixed with 4% PFA using purified ab52627 at a dilution of 1/150.

An Alexa Fluor^® 555 goat anti-rabbit was used as the secondary and the sample was stained with DAPI. An Alexa Fluor^® 555 goat anti-mouse was used at a dilution of 1/500 after ab52627 as the negative control and is shown in the bottom right hand panel.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gliocytoma tissue labelling SOX2 with unpurified ab92494 at 1/60. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

Formalin-fixed, paraffin-embedded human endometrium tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

ICC/IF image of ab105389 stained SKNSH cells. The cells were 4% formaldehyde 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 (ab105389, 1/200 dilution) 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.

All lanes : Anti-Nestin antibody [SP103] (ab105389) at 1/100 dilution

Lane 1 : Wild-type HAP1 whole cell lysate

Lane 2 : NES knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 177 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab105389 observed at 300 kDa. Red - loading control, ab18058, observed at 117 kDa.

ab105389 was shown to specifically react with NES in wild-type HAP1 cells as signal was lost in NES knockout cells. Wild-type and NES knockout samples were subjected to SDS-PAGE. Ab105389 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/100 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.