Recombinant Anti-Collagen I antibody [EPR7785] (ab138492)

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM/TBST.

Exposure Time: Lane 1-7: 180 seconds, Lane 8: 60 seconds.

ab181602 was used as loading control.

Compared with ab138492, ab255809 has higher affinity. We recommend ab255809 as an alternative for testing pro-Collagen forms in western blot.

ab138492 is specific for pro-Collagen and 139kda mature from, while ab255809 is specific for pro-Collagen and 35kda C-terminal pro peptide.

For better using ab255809, we recommend loading higher amount of lysate or using lower antibody dilution. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labeling Collagen I with purified ab138492 at 1/1500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody.

Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Blocking buffer: 5% NFDM/TBST.

Exposure time: 180 seconds.

The lysate in this image is prepared by 1%SDS Hot lysis method. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

IHC image of Collagen I staining in a section of frozen human cervix* performed on a Leica Biosystems BOND^® RX instrumen using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab138492, 0.01 ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Western blot results of Collagen I  from ADSCs (human adipose derived stem cells) cultured in RAD16-I alone, RAD/CS or RAD/Decorin. Actin was used as an internal control. Samples were prepared in triplicate; control, control medium; chondro, chondrogenic medium.

Samples were lysed in RIPA buffer with a protease inhibitor cocktail. Acrylamide gels were prepared according to the size of the proteins, generally at concentrations of 7.5% or 10% (w/v). Cell lysates (5 mg) were run by applying 150 V for 90 min. Proteins were transferred to a PVDF membrane by applying 40 V for 2 hours at RT. The membrane was incubated at RT for 2 hours in blocking buffer (BB) consisting of 4% (w/v) nonfat milk powder in PBST. Membranes were incubated for 1 hour at RT with ab138492 at a final concentration of 1 mg/mL in PBST. An anti-rabbit (IgG-HRP) secondary antibody was added, at a final concentration of 1 mg/mL, and incubated at RT for 1 h.

For full image please see paper.

IHC image of Collagen I staining in a section of frozen human uterus* performed on a Leica Biosystems BOND^® RX instrumen using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab138492, 0.05 ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Type I collagen (ab138492) and Type III collagen immunostainings for control, Subtype I, and Subtype II adenomyotic cases.

The type I collagen staining bands for adenomyotic cases were thicker than those of the control uteri, and were seen with more fine muscle bundles. Arrowheads indicate vascular walls. Original magnification: X100. Scale bar = 50μm.

Frozen, ground tissue samples were added to hot lysis buffer (10 mM Tris-HCl (pH 8.0); 1%SDS; 1.0 mM Na-Orthovanadate), boiled for 10-20 minutes and sonicated (3 seconds, 30 intervals).

The blocking and antibody incubations were performed in 5% non-fat milk (TBST).

The lysate in this image is prepared by 1%SDS Hot lysis method. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

Frozen, ground tissue samples were added to hot lysis buffer (10 mM Tris-HCl (pH 8.0); 1%SDS; 1.0 mM Na-Orthovanadate), boiled for 10-20 minutes and sonicated (3 seconds, 30 intervals).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

TBST).

The lysate in this image is prepared by 1%SDS Hot lysis method. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling Collagen I with unpurified ab138492 at 1/1500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody.

Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry of human breast carcinoma tissue staining Collagen I with ab138492 at 0.5μg/ml.

Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 was performed before commencing with IHC staining protocol.

Formalin/PFA-fixed paraffin-embedded sections of human breast carcinoma tissue stained for Collagen I with unpurified ab138492 in immunohistochemical analysis.

Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 was performed before commencing with IHC staining protocol.

Formalin/PFA-fixed paraffin-embedded sections of human colon tissue staining Collagen I with unpurified ab138492 in immunohistochemical analysis.

Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 was performed before commencing with IHC staining protocol.

IHC image of Collagen I staining in human placenta formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 minutes. The section was then incubated with ab138492 at 1/1500, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Paraffin-embedded human skin tissue stained for Collagen I using ab138492 at 1/3000 dilution in immunohistochemical analysis, followed by Goat anti rabbit Alexa Fluor­^® 555.

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