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Pro-Collagen I Antibody Staining Protocol for Immunohistochemistry
Description: Suitable for formaldehyde/formalin fixed paraffin sections as well as fresh frozen tissue sections. Does not stain mature collagen fibers in tissue. Localized intracellularly in cells producing pro-collagen I.
Primary Antibody
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Name: Pro-Collagen I Amino-Terminal Antibody |
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Clone: M-58, Rat anti-Human |
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Supplier: Chemicon |
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Catalog Number: MAB1912 |
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Dilution: 1:500 for paraffin sections, 1:10,000 for frozen sections using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| Heat/Cool Temperature: 37 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Collagen fibers |
| Images: Search image |
Additional Information:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections (no antigen retrieval needed) |
| Positive Control: Human skin |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
References:
1. Wright GM, Leblond CP (1981) Immunohistochemical localization of procollagens. III. Type I procollagen antigenicity in osteoblasts and prebone (osteoid). J Histochem Cytochem. 29(7):791-804. PubMed Abstract
2. Karim A, Cournil I, Leblond CP. (1979) Immunohistochemical localization of procollagens. II. Electron microscopic distribution of procollagen I antigenicity in the odontoblasts and predentin of rat incisor teeth by a direct method using peroxidase linked antibodies. J Histochem Cytochem. 27(7):1070-83. PubMed Abstract
3. Cournil I, Leblond CP, Pomponio J, Hand AR, Sederlof L, Martin GR. (1979) Immunohistochemical localization of procollagens. I. Light microscopic distribution of procollagen I, III and IV antigenicity in the rat incisor tooth by the indirect peroxidase-anti-peroxidase method. J Histochem Cytochem. 27(7):1059-69. PubMed Abstract