Immunohistochemistry analysis of Collagen I was performed on porcine skin tissue. To expose target proteins, antigen retrieval was performed by microwaving tissues for 8-15 minutes in 10mM sodium citrate buffer (pH 6.0). Following antigen retrieval, tissues were blocked in 3% hydrogen peroxide-methanol for 15 min at room temperature, washed with deionized water and PBS, and then probed with a Collagen I monoclonal antibody (Product # MA1-141) diluted 1:20 in 3% BSA-PBS (right panel) or incubated with buffer alone not containing primary antibody as a negative control (left panel), overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
|Tested species reactivity||Bovine, Human, Pig|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Purified, full-length native bovine collagen I protein|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||0.4 - 50 µg/ml|
|Immunohistochemistry (Frozen) (IHC (F))||1:200 - 1:1000|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10 - 1:100|
|Western Blot (WB)||1:500 - 1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
MA1-141 detects collagen I from bovine, human, and porcine. This antibody will not cross react with collagen 2-11, or detect thermally denatured collagen. Western blot analysis of recombinant bovine collagen I using MA1-141 detected a predominant band around 270kD likely corresponding to the alpha-1 dimer and a fainter band at a higher MW likely corresponding to the alpha-1/alpha-2 trimer.
MA1-141 has been successfully used in ELISA, immunofluorescence, immunohistochemistry (frozen and paraffin), and Western blot procedures. The epitope is sensitive to routine formalin fixation and paraffin embedding. Strong staining of connective tissue fibres is seen in acetone-fixed or unfixed frozen sections.
Type I collagen is a triple helix comprised of two alpha-1 chains and one alpha-2 chain. Type I collagen is a member of group I collagen (fibril-forming collagen) found in most connective tissues, and is abundant in bone, cornea, dermis and tendon. Mutations in the COL1A1 gene encoding the alpha-1 chain are associated with osteogenesis imperfecta types I-IV, Ehlers-Danlos syndrome type VIIA, Ehlers-Danlos syndrome Classical type, Caffey Disease and idiopathic osteoporosis. Mutations in the COL1A2 gene encoding the alpha-2 chain are associated with osteogenesis imperfecta types I-IV, Ehlers-Danlos syndrome type VIIB, recessive Ehlers-Danlos syndrome Classical type, idiopathic osteoporosis, and atypical Marfan syndrome. Symptoms associated with mutations in this gene, however, tend to be less severe than mutations in the COL1A1 gene, reflecting the different role of alpha-2 chains in matrix integrity.
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