Dab Staining Protocol

DAB Staining Protocol

DAB (3,3'-diaminobenzidine) is a chromogenic substrate that is used in immunohistochemistry and Western blotting to produce a brown precipitate at the site of antigen localization. DAB staining is a simple and sensitive method that can be used to visualize a wide range of antigens, including proteins, carbohydrates, and lipids.

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Materials

  • Tissue sections or Western blot membranes
  • Primary antibody
  • Secondary antibody conjugated to horseradish peroxidase (HRP)
  • DAB substrate kit
  • Hematoxylin (optional)

Procedure

  1. Deparaffinize and rehydrate tissue sections or soak Western blot membranes in PBS.
  2. Perform antigen retrieval (if necessary).
  3. Block tissue sections or membranes with blocking buffer (e.g., 10% BSA in PBS) for 30 minutes at room temperature.
  4. Incubate with primary antibody for 1-2 hours at room temperature or overnight at 4°C.
  5. Wash tissue sections or membranes with PBS 3 times for 5 minutes each.
  6. Incubate with secondary antibody conjugated to HRP for 1 hour at room temperature.
  7. Wash tissue sections or membranes with PBS 3 times for 5 minutes each.
  8. Prepare DAB substrate according to the manufacturer's instructions.
  9. Apply DAB substrate to tissue sections or membranes and incubate for 2-10 minutes, or until desired staining intensity is achieved.
  10. Rinse tissue sections or membranes with water.
  11. Counterstain tissue sections with hematoxylin (optional).
  12. Dehydrate and mount tissue sections or air-dry Western blot membranes.

Tips

  • For optimal results, use fresh DAB substrate.
  • DAB staining is sensitive to light, so it is important to perform the staining steps in a dark room or under red light.
  • Overstaining with DAB can result in a black precipitate. To avoid overstaining, monitor the staining process closely and stop the reaction when the desired staining intensity is achieved.
  • DAB staining can be combined with other immunohistochemical stains, such as fluorescent or metal-based stains, to produce multicolor images.

Applications

DAB staining is a versatile technique that can be used to visualize a wide range of antigens in a variety of tissues and cell cultures. DAB staining is commonly used in the following applications:

  • Immunohistochemistry: DAB staining is a standard method for detecting proteins and other antigens in tissue sections.
  • Western blotting: DAB staining can be used to detect proteins in Western blot membranes.
  • ELISA: DAB staining can be used to detect antigens in ELISA plates.
  • Flow cytometry: DAB staining can be used to detect antigens on cell surfaces.

DAB staining is a simple and sensitive method that is widely used in research and clinical settings. It is a valuable tool for studying the distribution and expression of antigens in tissues and cells.