One step Polymer HRP Mouse, Rat and Rabbit with DAB

Preparation of DAB Chromogen Reagent 5: To one ml of buffer substrate in a test tube, add two drops of reagent 5C mix well; this is ready-to-use DAB chromogen system. This reagent is good for 7-8 hours. Procedure: IHC/ICC procedure for frozen, paraffin sections and cell smears. 1. Deparafinize and hydrate tissue sections through xylene or other clearing agents and graded alcohols. 2. Rinse 2-3X with distilled or deionized water. 3. Incubate paraffin sections with Endoblocker for 10 minutes at room temperature. For frozen sections use Endoblocker. Rinse slide with distilled water 3X. 4. Note: If antigen retriever is required it can be applied at this step. Please refer to data sheet for the primary antibody. 5. Wash slide with PBS or Tris saline buffer or washing buffer 3X. 6. Incubate sections/ cell smear in Protein blocking solution, for 10 minutes at RT. Do not Rinse the slide. 7. Incubate sections/cell smear with primary antibody as recommended by the supplier. The primary antibody dilution buffer supplied can also be used as a negative control. 8. Wash slide with PBS/buffer 5-7X 9. Incubate with One-Step HRP polymer for 15 minutes at RT. 10. Wash slide 5-7 times with PBS/buffer. Caution: Peroxidase reagents are destroyed by sodium azide and should be avoided in all buffers and regents. 11. Wash slide with deionized or distilled for 2-3X. 12. Incubate with DAB chromogen reagent #5 for 5-10 minutes at RT; monitor the color development under microscope. 13. Wash slides 5-7X with distilled water. 14. Incubate with appropriate counterstain. 15. Wash slide with tap water, distilled water. 16. Mount slide with organic or aqueous mounting medium.