Name: Anti-Occludin antibody [EPR20992]
SKU: ab216327
Rating: 4 (7 reviews)

Anti-Occludin antibody [EPR20992] is a rabbit recombinant monoclonal antibody that is used to detect Occludin in Flow cytometry (Intra), ICC/IF, IHC-Fr, IHC-P, IP, Western blot. Suitable for Dog, Human, Mouse, Rat samples.

- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Specificity confirmed with OCLN knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR20992 is cited in over 330 publications

Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (AB216327), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	IHC-Fr	WB	ICC/IF	IHC-P	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested	Tested	Tested
Mouse	Expected	Expected	Tested	Not recommended	Tested	Expected
Rat	Expected	Expected	Tested	Not recommended	Tested	Expected
Dog	Expected	Expected	Tested	Tested	Expected	Expected
Recombinant full length protein - Human	Not recommended	Not recommended	Tested	Not recommended	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/40	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat, Dog	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/200	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat, Dog	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info 1/1000	Notes Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3. 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).
Species Mouse	Dilution info 1/1000	Notes Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3. 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). Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/1000	Notes Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3. 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).
Species Human	Dilution info 1/1000	Notes Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3. 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).
Species Dog	Dilution info 1/1000	Notes Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3. 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).

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes This antibody is not suitable for ICC in mouse or rat species.
Species Dog	Dilution info 1/100	Notes This antibody is not suitable for ICC in mouse or rat species.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes This antibody is not suitable for ICC in mouse or rat species.
Species Rat	Dilution info -	Notes This antibody is not suitable for ICC in mouse or rat species.
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/200	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/200	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/200	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Dog	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/60	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat, Dog	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Associated Products

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Target data

See full target information OCLN

Function

May play a role in the formation and regulation of the tight junction (TJ) paracellular permeability barrier. It is able to induce adhesion when expressed in cells lacking tight junctions. (Microbial infection) Acts as a coreceptor for hepatitis C virus (HCV) in hepatocytes.

Alternative names

Occludin, OCLN

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Clone number

EPR20992

Purification technique

Affinity purification Protein A

Specificity

This antibody is not suitable for ICC in mouse or rat species or for any testing rat intestinal tissues.

FURTHER INFORMATION ON SPECIFICITY (Chinese Version) available under the support & downloads section.

Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Anti-Occludin antibody [EPR20992] (ab216327) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), ICC/IF, IHC-Fr, IHC-P, IP and WB.

Anti-Occludin antibody [EPR20992] (ab216327) was first used in a scientific publication in 2018 and has been cited over 237 times in peer reviewed journals. It's performance in Western blot in human, mouse and rat samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-Occludin antibody [EPR20992] (ab216327) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-Occludin antibody [EPR20992] (ab216327) has been confirmed by Western Blot testing in Occludin knockout HAP1 cells.

Anti-Occludin antibody [EPR20992] (ab216327) has 8 independent reviews from customers.

Anti-Occludin antibody [EPR20992] (ab216327) specifically detects Occludin (UniProt ID: Q16625; Molecular weight: 60kDa) and is sold in a convenient trial size to enable initial testing (10 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).

Conjugation-ready, carrier free format available for antibody clone EPR20992 - Anti-Occludin antibody [EPR20992] - BSA and Azide free ab224526.

Occludin is a tight junction protein that plays a crucial role in maintaining the integrity of epithelial and endothelial barriers. In oncology, its downregulation is associated with increased tumorigenic and metastatic potential in cancers such as lung carcinoma, making it a potential prognostic biomarker and therapeutic target.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Occludin is a protein that plays an important mechanical role in forming tight junctions which are specialized connections between neighboring cell membranes. Alternative names for this target include occludin-1. The occludin protein has a molecular weight of approximately 65 kDa. It is expressed in a variety of tissues but levels tend to be high in epithelial and endothelial cells where tight junctions are essential for barrier functions. Occludin acts as integral membrane protein contributing to the complex structures that seal spaces between cells.

Biological function summary

Occludin serves as an important component in maintaining cell-to-cell adhesion within tight junction complexes. It interacts with other tight junction proteins such as claudins and junctional adhesion molecules to regulate paracellular permeability. These interactions help maintain selective barrier properties of epithelial and endothelial layers preventing the passage of large molecules and pathogens while allowing passage of ions and small molecules. Occludin stabilizes the tight junctions by interacting with the cytoskeleton and signaling proteins that are important for tight junction assembly and maintenance.

Pathways

Occludin functions prominently in the regulation of tight junction integrity and barrier function. It participates in pathways such as the tight junction signaling pathway and the paracellular transport pathway. Occludin interacts with ZO-1 a scaffolding protein bridging its interaction with the actin cytoskeleton which supports the structural organization and function of tight junctions. This connectivity allows for regulation of the permeability and transport across the epithelial barrier.

Associated diseases and disorders

Disruptions in occludin function are connected to conditions such as inflammatory bowel disease (IBD) and some cancers. In IBD abnormal occludin expression can compromise intestinal barrier function leading to increased intestinal permeability and an inflammatory response. In certain cancers loss of occludin expression is linked to increased invasiveness and metastasis due to weakened cell adhesion. The dysfunction of proteins like ZO-1 which interact with occludin in these pathways also contributes to these pathological states highlighting the importance of occludin's regulatory roles in maintaining cellular barrier integrity.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

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19 product images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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: Rabbit specific IHC polymer detection kit HRP/DAB 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.
Western blot - Anti-Occludin antibody [EPR20992] (ab216327)
Lanes 1 - 4: Merged signal (red and green). Green - ab216327 observed at 59 kDa. Red - loading control, Anti-GAPDH antibody [mAbcam 9484] - 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 Anti-GAPDH antibody [mAbcam 9484] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Occludin expression in HeLa is expected to be negative.
All lanes: Western blot - 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
Western blot - Anti-Occludin antibody [EPR20992] (ab216327)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-Occludin antibody [EPR20992] (ab216327) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution
Predicted band size: 59 kDa
Observed band size: 65 kDa
Exposure time: 60s
Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] (ab216327)
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) (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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.
Western blot - Anti-Occludin antibody [EPR20992] (ab216327)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-Occludin antibody [EPR20992] (ab216327) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution
Predicted band size: 59 kDa
Observed band size: 65 kDa
Exposure time: 100s
Immunoprecipitation - Anti-Occludin antibody [EPR20992] (ab216327)
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) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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).
All lanes: Immunoprecipitation - Anti-Occludin antibody [EPR20992] (ab216327)
Predicted band size: 59 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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: Rabbit specific IHC polymer detection kit HRP/DAB 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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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: Rabbit specific IHC polymer detection kit HRP/DAB 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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (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: Rabbit specific IHC polymer detection kit HRP/DAB 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.
Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] (ab216327)
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) (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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution.
Flow Cytometry (Intracellular) - Anti-Occludin antibody [EPR20992] (ab216327)
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 (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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (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 (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Counterstain was Hematoxylin.

Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
Western blot - Anti-Occludin antibody [EPR20992] (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
Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a GAPDH loading control.
All lanes: Western blot - Anti-Occludin antibody [EPR20992] (ab216327) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 16-60 kDa
Exposure time: 100s
Western blot - Anti-Occludin antibody [EPR20992] (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
Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a GAPDH loading control.
All lanes: Western blot - Anti-Occludin antibody [EPR20992] (ab216327) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 16-60 kDa
Exposure time: 20s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (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) (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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Rabbit specific IHC polymer detection kit HRP/DAB 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.
Immunohistochemistry (Frozen sections) - Anti-Occludin antibody [EPR20992] (ab216327)
Immunohistochemistry analysis of PFA-fixed, 0.3% Triton X-100 permeabilized frozen Human choroid plexus organoid (fresh frozen) tissue labelling Occludin with ab216327 at 1/200 dilution, followed by Donkey anti Rabbit (Alexa Fluor® 488) secondary antibody at 1/500 dilution dilution (green). The section was incubated with ab216327 for 24 hours at 4°C.