Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab315228 at a 1/2000 (0.263 μg/ml) dilution, Collagen I with ab270993 and Langerin with 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 ab315228, ab270993 and 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab270993 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using ab270993, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse pancreatic cancer tissue labeling Collagen I with 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 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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab270993 at 1/2000 dilution, followed by 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. 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 ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab278525 at a 1/5000 (0.109 μg/ml) dilution, PAX8 with ab239363 at 1/2000 (0.254 μg/ml) dilution, and Collagen I with 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 ab278525, ab239363 and 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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab270993 at 1/100 dilution followed by 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 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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using ab270993, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat skin tissue labeling Collagen I with 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 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using ab270993, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Collagen I with 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 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using ab270993, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labeling Collagen I with 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 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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab270993 at 1/500 dilution followed by 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 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).

• IP

Supplier Data

Immunoprecipitation - Anti-Collagen I antibody [EPR24331-53] - BSA and Azide free (AB279711)

This data was developed using 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 ab270993 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270993 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse skin tissue lysate 10 ug

Lane 2 : ab270993 IP in Mouse skin tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of 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] (<a href='/en-us/products/primary-antibodies/collagen-i-antibody-epr24331-53-ab270993'>ab270993</a>)

Predicted band size: 139 kDa

Observed band size: 138 kDa

false

• WB

Supplier Data

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. ab181602 was used as a loading control. This data was developed using ab270993, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Collagen I antibody [EPR24331-53] (<a href='/en-us/products/primary-antibodies/collagen-i-antibody-epr24331-53-ab270993'>ab270993</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 139 kDa

Observed band size: 139 kDa

false

Exposure time: 100s

• WB

Lab

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

This data was developed using 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] (<a href='/en-us/products/primary-antibodies/collagen-i-antibody-epr24331-53-ab270993'>ab270993</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 139 kDa

Observed band size: 220 kDa

false

Exposure time: 20s

• WB

Lab

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

This data was developed using 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] (<a href='/en-us/products/primary-antibodies/collagen-i-antibody-epr24331-53-ab270993'>ab270993</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 139 kDa

Observed band size: 138 kDa

false