Name: Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free
SKU: ab279711
Rating: 3 (1 reviews)

Rabbit Recombinant Monoclonal COL1A1 antibody. Carrier free. Suitable for ICC/IF, IP, WB, IHC-Fr, Flow Cyt (Intra), IHC-P, mIHC and reacts with Mouse, Rat samples. Cited in 3 publications.

Related conjugates and formulations

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Alexa Fluor® 750 Anti-Collagen I antibody [EPR24331-53]
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Alexa Fluor® 555 Anti-Collagen I antibody [EPR24331-53]
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Alexa Fluor® 594 Anti-Collagen I antibody [EPR24331-53]
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Alexa Fluor® 488 Anti-Collagen I antibody [EPR24331-53]
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Alexa Fluor® 647 Anti-Collagen I antibody [EPR24331-53]

Images

Western blot - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Constituents: 100% PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

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

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ICC/IF	IP	WB	IHC-Fr	Flow Cyt (Intra)	IHC-P	mIHC
Human	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended
Mouse	Tested	Tested	Tested	Tested	Tested	Tested	Tested
Rat	Expected	Expected	Tested	Tested	Expected	Tested	Expected

Tested
Tested

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes -

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes For mouse samples, please use high sensitivity substrate.
Species Rat	Dilution info -	Notes For mouse samples, please use high sensitivity substrate.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes For mouse samples, please use high sensitivity substrate.

Tested
Tested

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)
Species Mouse	Dilution info -	Notes Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes -

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

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

Not recommended
Not recommended

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

Tested
Tested

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Associated Products

Select an associated product type

6 products for Alternative Version

Target data

See full target information Col1a1

Function

Type I collagen is a member of group I collagen (fibrillar forming collagen).

Alternative names

Cola1, Col1a1, Collagen alpha-1(I) chain, Alpha-1 type I collagen

Recommended products

Rabbit Recombinant Monoclonal COL1A1 antibody. Carrier free. Suitable for ICC/IF, IP, WB, IHC-Fr, Flow Cyt (Intra), IHC-P, mIHC and reacts with Mouse, Rat samples. Cited in 3 publications.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Constituents: 100% PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Carrier free: Yes
Clone number: EPR24331-53
Purification technique: Affinity purification Protein A
Specificity: FURTHER INFORMATION ON SPECIFICITY (Chinese Version) available under the support & downloads section.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: +4°C

Notes

ab279711 is the carrier-free version of Anti-Collagen I antibody [EPR24331-53] ab270993.

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.

Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

Supplementary info

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

Activity summary

Collagen type I also called collagen I is a structural protein expressed mainly in connective tissues such as skin tendon bone and ligaments. It serves as an important component in providing mechanical strength and integrity to these tissues. Collagen I is a fibrillar collagen known for its triple-helix structure composed of two alpha-1 chains and one alpha-2 chain and has a molecular mass of approximately 300 kDa. Researchers often employ collagen western blot and collagen ELISA techniques for its detection. Collagen suppliers offer various collagen antibodies used in these assays to study its distribution and function.

Biological function summary

Collagen type I plays a central role in maintaining the extracellular matrix and supporting cellular environments. It interacts with other matrix proteins and cells forming complexes that help in tissue development and repair. Type I collagen is especially important in bone matrix working alongside minerals like hydroxyapatite to provide rigidity and support. Anti-collagen antibodies aid in studying its biological functions and interactions which are critical to understanding tissue dynamics.

Pathways

Collagen type I interacts with multiple signaling cascades involved in tissue remodeling and repair. It is a significant player in the TGF-β pathway which regulates fibrosis and wound healing processes. In these pathways proteins such as fibronectin and integrins work in concert with collagen type I to orchestrate cellular responses to damage. Researchers often examine its role in these pathways to uncover therapeutic possibilities for disease interventions.

Associated diseases and disorders

Collagen type I has strong connections to conditions like osteogenesis imperfecta and fibrosis. Mutations or irregularities in collagen I production can lead to osteogenesis imperfecta a genetic disorder characterized by brittle bones. In fibrosis excessive collagen deposition disrupts normal tissue architecture contributing to organ dysfunction. In both conditions type I collagen interacts with other proteins like matrix metalloproteinases which modulate its breakdown and remodeling highlighting its importance in disease pathology.

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.

Full details and terms and conditions can be found here:
Terms & Conditions.

14 product images

Western blot - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Blocking buffer and concentration: 5% NFDM/TBST
Diluting buffer and concentration: 5% NFDM/TBST
We are unsure how to define these extra bands below 100kDa.
The molecular weight observed is consistent with what has been described in the literature (PMID:23940311;PMID:29853175).
All lanes: Western blot - Anti-Collagen I antibody [EPR24331-53] (Anti-Collagen I antibody [EPR24331-53] ab270993) at 1/1000 dilution
All lanes: NIH/3T3 (mouse embryonic fibroblast), 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
Predicted band size: 139 kDa
Observed band size: 220 kDa
Exposure time: 20s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in connective tissues of mouse skin. The section was incubated with Anti-Collagen I antibody [EPR24331-53] ab270993 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunoprecipitation - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Collagen I was immunoprecipitated from 0.35 mg Mouse skin tissue lysate 10 ug with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: Mouse skin tissue lysate 10 ug
Lane 2: Anti-Collagen I antibody [EPR24331-53] ab270993 IP in Mouse skin tissue lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-Collagen I antibody [EPR24331-53] ab270993 in Mouse skin tissue lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds
All lanes: Immunoprecipitation - Anti-Collagen I antibody [EPR24331-53] (Anti-Collagen I antibody [EPR24331-53] ab270993)
Predicted band size: 139 kDa
Observed band size: 138 kDa
Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cells is observed. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse skin tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on mouse skin is observed. The nuclear counterstain was DAPI (Blue).
Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
Flow Cytometry (Intracellular) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (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-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in connective tissues of mouse stomach. The section was incubated with Anti-Collagen I antibody [EPR24331-53] ab270993 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded Mouse pancreatic cancer tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in connective tissues of mouse pancreatic cancer. The section was incubated with Anti-Collagen I antibody [EPR24331-53] ab270993 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Western blot - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Blocking and diluting buffer and concentration: 5% NFDM/TBST
The observed MW is consistent with what has been described in the literature (PMID: 27740527;PMID: 22278938; PMID: 26973392).
Exposure time: Lane 1: 3.25 seconds
Lane 2: 5.5 seconds
All lanes: Western blot - Anti-Collagen I antibody [EPR24331-53] (Anti-Collagen I antibody [EPR24331-53] ab270993) at 1/1000 dilution
Lane 1: Mouse skin tissue lysate at 20 µg
Lane 2: Rat skin 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 1/100000 dilution
Predicted band size: 139 kDa
Observed band size: 138 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded Rat skin tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in connective tissues of rat skin. The section was incubated with Anti-Collagen I antibody [EPR24331-53] ab270993 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat skin tissue labeling Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on rat skin is observed. The nuclear counterstain was DAPI (Blue).
Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
Western blot - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM/TBST.

This blot was produced using a high sensitivity ECL substrate.

Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a loading control.

This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
All lanes: Western blot - Anti-Collagen I antibody [EPR24331-53] (Anti-Collagen I antibody [EPR24331-53] ab270993) at 1/1000 dilution
Lane 1: Mouse skin tissue lysate at 20 µg
Lane 2: Mouse kidney tissue lysate at 20 µg
Lane 3: Mouse lung tissue lysate at 20 µg
Lane 4: Mouse heart 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
Predicted band size: 139 kDa
Observed band size: 139 kDa
Exposure time: 100s
Multiplex immunohistochemistry - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse skin staining of Cytokeratin 1 + Cytokeratin 2e with Anti-Cytokeratin 1 + Cytokeratin 2e antibody [EPR27257-91] ab315228 at a 1/2000 (0.263 μg/ml) dilution, Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 and Langerin with Anti-Langerin antibody [EPR24685-12] ab283686 at a 1/1000 dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Cytokeratin 1 + Cytokeratin 2e (green; Opal™520), anti-Collagen I (magenta; Opal™690) and anti-Langerin (gray; Opal™570) on mouse skin.

Panel B: anti-Cytokeratin 1 + Cytokeratin 2e staining epidermis keratin layer in mouse skin.

Panel C: anti-Collagen I staining connective tissues in mouse skin.

Panel D: anti-Langerin staining Langerhans cells in mouse skin.

Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Cytokeratin 1 + Cytokeratin 2e antibody [EPR27257-91] ab315228, Anti-Collagen I antibody [EPR24331-53] ab270993 and Anti-Langerin antibody [EPR24685-12] ab283686 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system,
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Multiplex immunohistochemistry - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (ab279711)
This data was developed using Anti-Collagen I antibody [EPR24331-53] ab270993, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse thyroid staining of Thyroid Peroxidase/TPO with Anti-Thyroid Peroxidase/TPO antibody [EPR23574-405] ab278525 at a 1/5000 (0.109 μg/ml) dilution, PAX8 with Anti-PAX8 antibody [EPR23508-20] ab239363 at 1/2000 (0.254 μg/ml) dilution, and Collagen I with Anti-Collagen I antibody [EPR24331-53] ab270993 at a 1/500 (1.152 μg/ml) dilution, followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.
Panel A: merged staining of anti-Thyroid peroxidase (green; Opal™ 520), anti-PAX8 (magenta; Opal™ 690) and anti-Collagen I (gray; Opal™ 570) on mouse thyroid.

Panel B: anti-Thyroid peroxidase showed cytoplasmic and membranous staining on mouse thyroid.

Panel C: anti-PAX8 showed nucleus staining on mouse thyroid.

Panel D: anti-Collagen I staining connective tissues in mouse thyroid.

Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-Thyroid Peroxidase/TPO antibody [EPR23574-405] ab278525, Anti-PAX8 antibody [EPR23508-20] ab239363 and Anti-Collagen I antibody [EPR24331-53] ab270993 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.