Anti-Collagen I antibody [EPR24331-53]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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] (AB270993)

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-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] (AB270993)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse skin labelling Collagen I with ab270993 at a concentration of 0.025µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins.

ab270993 anti-Collagen I antibody [EPR24331-53] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR24331-53] (AB270993)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse skin labelling Collagen I with ab270993 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab270993 anti-Collagen I antibody [EPR24331-53] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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] (AB270993)

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] (AB270993)

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] (AB270993)

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] (AB270993)

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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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.

• IP

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Immunoprecipitation - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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] (ab270993)

Predicted band size: 139 kDa

Observed band size: 138 kDa

false

• WB

Lab

Western blot - Anti-Collagen I antibody [EPR24331-53] (AB270993)

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] (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) (<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] (AB270993)

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] (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) (<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

• WB

Supplier Data

Western blot - Anti-Collagen I antibody [EPR24331-53] (AB270993)

Blocking buffer and concentration : 5% NFDM/TBST. Diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as a loading control. This blot was produced using a high sensitivity ECL substrate.

All lanes:

Western blot - 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) (<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