Application Notes: Further information: Dilute antibody to its recommended working dilution using the same buffer defined in the Presentation. Manual Use: 1. Pretreatment: Perform heat-induced epitope retrieval (HIER) at pH 9 for 10 to 30 minutes. 2. Peroxide Block: Block in peroxidase blocking solution for 5 minutes at room temperature. (Not required if using Alkaline Phosphatase System.) 3. Primary Antibody: Dilute antibody at 1:100-1:200 before applying. Incubate antibody for 10 to 30 minutes at room temperature. 4. Secondary Antibody: Incubate for 20 to 30 minutes at room temperature. 5. Substrate Development: Incubate DAB or Fast Red for 5 to 10 minutes at room temperature. 6. Counterstain: Counterstain with hematoxylin for 0.5 to 5 minutes, depending on the hematoxylin used. Rinse with distilled water and blueing solution for 30 seconds. 7. Dehydrate and apply coverslip
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