Anti-Collagen I antibody [RM1131]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human breast cancer.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed 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 [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human endometrium.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed 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 [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human colon.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [RM1131] (AB316222)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HFF-1 (human skin fibroblast) cells labelling Collagen I with ab316222 at 1/100 (5.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in HFF-1 cell line.
Negative control : HT-29.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/500 (1ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human stomach.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Collagen I antibody [RM1131] (AB316222)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HT-29 (human colorectal adenocarcinoma epithelial cell, Left) / HFF-1 (human skin fibroblast, Right) cells labelling Collagen I with ab316222 at 1/5000 dilution (0.01 ug)/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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : HT-29.

• IP

Supplier Data

Immunoprecipitation - Anti-Collagen I antibody [RM1131] (AB316222)

Collagen I was immunoprecipitated from 0.35 mg HFF-1 (human skin fibroblast) whole cell lysate with ab316222 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316222 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HFF-1 (human skin fibroblast) whole cell lysate
Lane 2 : ab316222 IP in HFF-1 (human skin fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316222 in HFF-1 whole cell lysate

All lanes:

Immunoprecipitation - Anti-Collagen I antibody [RM1131] (ab316222) at 1/30 dilution

All lanes:

HFF-1 (human skin fibroblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse skin (fresh frozen) tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on mouse skin. The nuclear counterstain was DAPI (Blue). The section was incubated with ab316222 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Collagen I antibody [RM1131] (AB316222)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell, Left) / L6 (rat skeletal muscle myoblast, Right) cells labelling Collagen I with ab316222 at 1/5000 dilution (0.01 ug)/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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : C6.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Rat stomach tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of rat stomach.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed 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 [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of mouse skin.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat skin (fresh frozen) tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on rat skin. The nuclear counterstain was DAPI (Blue). The section was incubated with ab316222 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [RM1131] (AB316222)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized L6 (rat skeletal muscle myoblast) cells labelling Collagen I with ab316222 at 1/100 (5.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in L6 cell line.
Negative control : C6.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/500 (1ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [RM1131] (AB316222)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Collagen I with ab316222 at 1/100 (5.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in NIH/3T3 cell line.
Negative control : RAW 264.7.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/500 (1ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Collagen I antibody [RM1131] (AB316222)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage, Left) / NIH/3T3 (mouse embryonic fibroblast, Right) cells labelling Collagen I with ab316222 at 1/5000 dilution (0.01 ug)/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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : Raw 264.7.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of mouse stomach.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed 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 [RM1131] (AB316222)

Immunohistochemical analysis of paraffin-embedded Rat skin tissue labeling Collagen I with ab316222 at 1/500 (1.012 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of rat skin.
The section was incubated with ab316222 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Collagen I antibody [RM1131] (AB316222)

Collagen I was immunoprecipitated from 0.35 mg Mouse skin tissue lysate with ab316222 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316222 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse skin tissue lysate
Lane 2 : ab316222 IP in Mouse skin tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316222 in mouse skin tissue lysate

All lanes:

Immunoprecipitation - Anti-Collagen I antibody [RM1131] (ab316222) at 1/30 dilution

All lanes:

Mouse skin tissue lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

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

Negative control : HT-29.
We are unsure how to define these extra bands below 100kDa.

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/1000 dilution

Lane 1:

HFF-1 (human skin fibroblast) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) culture supernatant at 10 µL

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 220 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

The molecular weight observed is consistent with what has been described in the literature (PMID : 23940311)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1 : 180 seconds, Lane 2 : 8 seconds, Lane 3 : 81 seconds

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/5000 dilution

Lane 1:

Mouse skin tissue lysate at 20 µg

Lane 2:

Rat skin tissue lysate at 20 µg

Lane 3:

Human uterus tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 139 kDa,220 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

Collagen I might be easily degraded. Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/1000 dilution

Lane 1:

HFF-1 (human skin fibroblast) whole cell lysate at 20 µg

Lane 2:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 220 kDa,36 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

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

This blot was developed using a high sensitivity ECL substrate.

Low expression tissue : normal mouse kidney (PMID : 33758176, PMID : 29102563)

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/1000 dilution

Lane 1:

Mouse skin tissue lysate at 20 µg

Lanes 2 and 5:

Mouse kidney tissue lysate at 20 µg

Lanes 3 and 6:

Mouse lung tissue lysate at 20 µg

Lanes 4 and 8:

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: 180s

• WB

Lab

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

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

Negative control : RAW 264.7.
We are unsure how to define these extra bands below 100kDa.

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/5000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) culture supernatant at 10 µL

Lane 3:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 220 kDa

false

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-Collagen I antibody [RM1131] (AB316222)

Negative control : C6.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Collagen I antibody [RM1131] (ab316222) at 1/1000 dilution

Lane 1:

L6 (rat skeletal muscle myoblast) whole cell lysate at 20 µg

Lane 2:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 220 kDa,36 kDa

false

Exposure time: 10s