Anti-Occludin antibody [EPR20992] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

Immunohistochemical analysis of paraffin-embedded human breast 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 breast is observed. Counter stained with Hematoxylin.

Secondary antibody only control : ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

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).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327). Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880), ready to use. Counter stained with Hematoxylin. Antigen retrieval was heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Secondary antibody only control : ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB). Positive staining on human thyroid carcinoma. The section was incubated with ab216327 at 4°C overnight.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Immunohistochemical analysis of paraffin-embedded Human colon labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Positive staining on human colon. The section was incubated with ab216327 at 4°C overnight. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using ab216327, the same antibody clone in a different buffer formulation.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

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 : ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cell line labeling Occludin with ab216327 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

• IP

Supplier Data

Immunoprecipitation - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Occludin was immunoprecipitated from 0.35 mg of Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate with ab216327 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab216327 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Caco-2 whole cell lysate 10 μg (Input).

Lane 2 : ab216327 IP in Caco-2 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab216327 in Caco-2 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

The molecular weight observed is consistent with what has been described in the literature (PMID : 18647175, PMID : 19821483).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

All lanes:

Immunoprecipitation - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>)

Predicted band size: 59 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Immunohistochemical analysis of paraffin-embedded Rat colon labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Positive staining on Rat colon. The section was incubated with ab216327 at 4°C overnight. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using ab216327, the same antibody clone in a different buffer formulation.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

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 : ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Immunohistochemical analysis of paraffin-embedded rat 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 rat kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control : ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Immunohistochemical analysis of paraffin-embedded Mouse colon labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Positive staining on Mouse colon. The section was incubated with ab216327 at 4°C overnight. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using ab216327, the same antibody clone in a different buffer formulation.

• WB

Lab

Western blot - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

All lanes:

Western blot - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>) 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

false

• WB

Lab

Western blot - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

For cells express relatively low level of Occludin, we suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results.

Blocking and Diluting buffer and concentration : 5% NFDM /TBST

ab181602 was used as a GAPDH loading control.

All lanes:

Western blot - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>) at 1/1000 dilution

Lane 1:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

DLD-1(human colorectal adenocarcinoma cell) whole cell lysate at 20 µg

Lane 3:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 16-60 kDa

false

Exposure time: 20s

• WB

Unknown

Western blot - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using ab216327, the same antibody clone in a different buffer formulation.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>) at 1/1000 dilution

Lane 1:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

bEnd.3 (Mouse brain endothelioma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 59 kDa

Observed band size: 65 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

Occludin contains multiple isoforms, and the molecular weight range of different isoforms varies greatly (8kDa~59kDa). In addition, there are various post-translational modifications such as ubiquitination and proteolytic cleavage, so the band sizes and number of bands detected in different samples may vary. (PMID : 18647175, PMID : 19821483, PMID : 19457074, PMID : 11782481, PMID : 22083955)

Blocking and Diluting buffer and concentration : 5% NFDM /TBST

ab181602 was used as a GAPDH loading control.

All lanes:

Western blot - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>) at 1/1000 dilution

Lane 1:

Mouse colon tissue lysate at 20 µg

Lane 2:

Mouse small intestine tissue lysate at 20 µg

Lane 3:

Mouse ileum tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Mouse Kidney tissue lysate at 20 µg

Lane 6:

Human colon tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 16-60 kDa

false

Exposure time: 100s

• WB

Unknown

Western blot - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using ab216327, the same antibody clone in a different buffer formulation.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Occludin antibody [EPR20992] (<a href='/en-us/products/primary-antibodies/occludin-antibody-epr20992-ab216327'>ab216327</a>) at 1/1000 dilution

Lane 1:

Mouse colon tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse lung tissue lysate at 20 µg

Lane 4:

Rat colon tissue lysate at 20 µg

Lane 5:

Rat brain tissue lysate at 20 µg

Lane 6:

Rat lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 59 kDa

Observed band size: 65 kDa

false

Exposure time: 100s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDCK (canine kidney 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 MDCK (NBL-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).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (AB224526)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327). Tissue Microarrays stained for Anti-Occludin antibody [EPR20992] using ab216327 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The sections were incubated with ab216327 at 4°C overnight used at 1 : 2000 dilution (1.05 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB secondary antibody (ab209101). Counterstain was Hematoxylin. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).