Anti-Collagen I antibody (ab34710)

Other Band(s): Collagen Type I splice variants and isoforms.

The expression of Collagen I was examined in Normal fibroblasts and keloid fibroblasts using ab34710 at 1/50 dilution in ICC/IF analysis.

Cells were cultured on microscope coverslips (Thermo Fisher Scientific, Rochester, NY, USA) and treated with NTP (Non-thermal plasma). After 24 h, the slides were washed with PBS, fixed for 20 min in 3.7% formaldehyde and rehydrated in PBS. After blocking for 45 min in bovine serum albumin dissolved in 5% PBS, the slides were incubated for 1 h with sb34710 at 1/50 dilution  then washed with PBS and incubated for 45 min with an Alexa 488-labeled goat anti-rabbit antibody at 1/250 dilution.

NTP reduced the expression of Type I collagen in KFs as revealed by the immunocytochemistry and Sirus Red stain. Immunofluorescent and Sirus Red staining for Type I collagen were observed in NFs and KFs after treatments with gas or NTP.

Row 1 and 2: Analysis of Collagen I in Normal fibroblasts by Sirus Red and IF respectively.
Row 3 and 4: Analysis of Collagen I in Keloid fibroblasts by Sirus Red and IF respectively.

Column 1 is the control. Column 2 is the control gas treatment (Argon and nitrogen at 200:500). Column 3-5 were exposed to NTP for 10, 30 and 60 seconds respectively.

ab34710 staining Collagen I in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2.5% horse serum for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500 in PBS 1X + 3% of 2.5% horse serum) for 12 hours at 4°C. An undiluted HRP-conjugated Horse anti-rabbit polyclonal was used as the secondary antibody.

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ab34710 staining Collagen I in pig lung tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with 10% buffered formalin and blocked with 5% serum for 1 hour at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500) for 12 hours at 4°C. A Cy3-conjugated Donkey anti-rabbit polyclonal (1/200) was used as the secondary antibody.

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ab34710 staining Collagen I in horse bronchial fibroblast cells by Immunocytochemistry. Cells were fixed with acetone and blocking with 3% BSA was performed for 1 hour 30 minutes at 40°C. Samples were incubated with primary antibody (1/500: in 3% BSA/ PBS) for 12 hours at 4°C. An FITC-conjugated goat polyclonal to rabbit IgG was used at dilution at 1/160 as secondary antibody.

ab34710 staining Collagen I in sheep jejunum tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 0.25% protein block for 1 hour at room temperature; antigen retrieval was by heat mediation in a 10 mM citrate buffer, pH 6. Samples were incubated with primary antibody (1/400) for 1 hour. An Envision +system HRP anti-rabbit, goat polyclonal (HRP polymer conjugation) was used as the secondary antibody.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of marmoset kidney tissue labeling Collagen I with ab34710 at 1/500 dilution. Tissue samples were fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C. Heat mediated antigen retirval was performed using citric acid. Samples were incubated with primary antibody (1/500 in TBS/BSA/azide buffer) for 2 hours at 21°C. A biotin conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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IHC image of Collagen I staining in normal human placenta formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab34710 at 5 µg/ml, 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

ab34710 staining Collagen I in cow small intestine tissue sections by Immunohistochemistry (Formalin/PFA-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1000 in TBS/BSA/azide buffer) for 2 hours at 21°C. A biotin conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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ab34710 staining mouse kidney sections (ab4606) by IHC-Fr. Kidney tissue was cryoprotected with 30% sucrose, sectioned using a cryostat at 10 microns and mounted onto slides. After drying overnight in fume hood, sections were fixed in 4% formalin in PBS for 10 minutes. Blocking was performed with 1% BSA for 10 minutes at 21°C. Staining with ab34710 at a 1/100 dilution in TBS/BSA with 0.02% azide was performed for 2 hours at 21°C. A conjugated goat anti-rabbit 594 polyclonal antibody at 1/1000 was used as the secondary antibody.

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Detection of collagen I in Wistar rat hepatic stellate cells in GFP-transduced (left lane) and PPAR?-transduced cell lysates (right lane). Protein staining shown below each blot depicts equal protein loading. An equal amount of the whole cell protein (100 µg) was separated by native PAGE and electroblotted to nitrocellulose membranes. Proteins were detected by incubating the membrane with anti-Collagen I antibody at a concentration of 0.2–2 µg/10 ml in TBS with 5% non-fat milk.

Immunohistochemistry of breast carcinoma staining Collagen I with ab34710 at 5μg/ml

Immunohistochemical analysis of human colon tissue sections labelling Collagen I with ab34710 at a dilution of 1/200. The sections were fixed with formaldehyde. The secondary antibody used was an HRP conjugated rabbit IgG. Antigen retrieval was heat mediated using CC1.

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Immunohistochemical analysis of formaldehyde-fixed frozen bovine tendon sections, labelling Collagen I with ab34710 at a dilution of 1/500 for 2 hours at 21°C. 1% BSA was used as a blocking agent and incubated for 10 minutes at 21°C. Secondary used was a Goat anti-Rabbit Alexa Fluor^® 594 conjugate used at 1/1,000.

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