Concentrated HRP Ant Rabbit IgG (H+L) kit
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Concentrated HRP Ant Rabbit IgG (H+L) kit; 10X Catalog number: IH-8112 A. Reagents Provided: The following reagents are 10X concentrated, when diluted makes 50 ml of working solution. For Immunohistochemistry (IHC) can yield 500 tests, when 0.1 ml is used per slide. Bottle # Quantity Description Reagent A 10 ml Mixing reagent (pink color cap) Reagent B 5 ml 10X Peroxidase block (white color cap) Reagent C 5 ml 10X Protein blocking solution (blue color cap) Reagent D 5 ml 10X Biotinylated anti-Rabbit IgG (H+L) (yellow color cap) Reagent E 5 ml 10X Streptavidin conjugated to peroxidase (HRP) (orange color cap) B. Reagents Required but not supplied: Washing buffer, antigen retrievers, positive or negative control, primary antibodies, primary antibody dilution buffer, chromogen, counterstain and mounting medium. Description: Immunohistochemistry (IHC)./ Immunocytochemistry (ICC) is the localization of antigens by the use of antigens in tissue sections/cells by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. Several IHC techniques are commonly used: labeled biotin secondary antibody streptavidin-peroxidase (LBSASP), HRP anti-HRP, ABC, catalyzed signal amplification, polymer system and others, to detect antigens on tissue and cell In this kit the first layer is unlabeled primary antibody, the second layer is biotinylated secondary antibody, the third layer is Enzyme-Streptavidin conjugate (HRP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solution to produce different colorimetric end products. Intended Use: Immunohistochemistry (IHC) and Immunocytchemistry (ICC). (This kit can be used for WB or ELISA; the dilution should be determined by the individual lab. Normally for WB the IHC reagents are diluted 2-5X and for ELISA the IHC reagents are diluted 10-100 X. For ELISA, one has to use soluble chromogen, like TMB).The optimum dilutions for WB or ELISA should be determined by the individual lab. Dilution of Reagents: Preparation of Working Peroxidase solution (Working Solution B): To one ml of distilled or deionized water add 3 drops of reagent B. Preparation of working protein blocking solution (Working Solution C): To one ml of distilled or deionized water add 3 drops of mixing reagent A and 3 drops of reagent C. Preparation of working Biotinylated secondary antibody (Working Solution D): To one ml of distilled or deionized water, add 3 drops of mixing reagent A and 3 drops of reagent D. Preparation of working streptavidin peroxidase reagent (Working Solution E) To one ml of distilled or deionized water , add 3 drops of mixing reagent A and 3 drops of reagent E These ready to use solutions can be refrigerated and are good for few days. Storage: 2-8°C Procedure: IHC/ICC procedure for frozen sections, paraffin sections and cell smears. 1. Deparafinize and hydrate tissue sections through xylene or other clearing agents and graded alcohols.(For frozen sections or cell smears; use unfixed, acetone fixed or appropriate fixative for the antigen in question; for cell smears it may be necessary to permealize the cell by detergent, please refer to antibody protocol) 2. Wash 2-3 with distilled or deionized water. 3. Incubate sections/cell smear in Peroxidase block (Working solution B) for 5-10 minutes at room temperature or 37°C. Note: If antigen retriever is required it can be applied after this stage. 4. Wash slide with PBS Tris saline (with 0.02-0.05% nonionic detergent, Triton X100, Tween 20 or NP-40) or washing buffer (Universal Immunoassay buffer IBSC cat # AR-6561) 3-5X. 5. Incubate sections/ cell smear in Protein blocking solution (Working solution C) for 10 minutes. at RT or 37ºC 6. Wash slide with PBS 1X. 7. Incubate sections/cell smear in primary antibody for 20-30 minutes at room temperature or 37°C. (For more information, refer to instructions for primary antibody) 8. Wash slide with PBS 5-7X 9. Incubate with biotinylated secondary antibody (Working solution D) for 15 minutes at room temp. or 37°C. 10. Wash slide 5-7 times with buffer. Caution: Peroxidase reagents are destroyed by sodium azide and should be avoided in all buffers and regents. 11. Incubate with Streptavidin-Peroxidase reagent (Working solution E) for 10 minutes at room temperature or 37ºC. 12. Wash slide with PBS for 5-7 X. 13. Wash slide with deionized or distilled for 2-3X. 14. Incubate with AEC reagent or DAB or other chromogen (NOT SUPPLIED) for 5-10 minutes at room temperature or 37ºC. 15. Wash slide with distilled or deionized water 5-7X. 16. Incubate with proper counterstain (NOT SUPPLIED) 30-60 seconds or longer. 17. Wash slide with tap water, distilled water, followed by PBS buffer. 18. Wash slide with distilled or deionized water. Now this slide is ready to be mounted with mounting medium. Please see instructions for chromogen, counterstain and mounting medium. These are guide lines, the optimum incubation times for these reagents and reactions should be determined by the individual lab. --------------------------------------------------------------------------------------------------------- Limitation and warranty: Our warranty is limited to the actual price paid for the product. We are not liable for any property damage, personnel injury, time, effort or economic loss due to our product. MSDS: Avoid skin and eye contact with all laboratory products. Use appropriate lab. gear, lab coat , gloves and safety glasses. Do not ingest any lab. products. This product is not approved for administration in human or animals. --------------------------------------------------------------------------------------------------------------------------------- |
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