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.

All lanes : Anti-Collagen I antibody [EPR7785] (ab138492) at 1/5000 dilution

Lane 1 : HFF-1 (human skin fibroblast) whole cell lysate
Lane 2 : MRC-5 (Human lung fibroblast) whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 139 kDa
Observed band size: 220 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST.

Exposure time: 180 seconds.

All lanes : Anti-Collagen I antibody [EPR7785] (ab138492) at 1/1000 dilution (unpurified)

Lane 1 : Human stomach tissue lysate
Lane 2 : Human skin lysate
Lane 3 : Human adrenal gland lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution
Lanes 2-3 : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 139 kDa

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).

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.

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.

Anti-Collagen I antibody [EPR7785] (ab138492) at 1/5000 dilution (unpurified) + Human skin tissue lysate at 10 µg

Secondary
HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 139 kDa
Observed band size: 139 kDa

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).

Anti-Collagen I antibody [EPR7785] (ab138492) at 1/5000 dilution (purified) + Human skin tissue lysate at 10 µg

Secondary
HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/5000 dilution

Predicted band size: 139 kDa
Observed band size: 139 kDa

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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