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How to Perform Peroxidase Stains

Detection of antibodies is important for both laboratory research and clinical diagnostics. Normally, antibodies are invisible. If you want to detect antibodies in a piece of tissue, you must use a strategy other than merely looking through a microscope. Alkaline phosphatase staining and peroxidase staining are two such methods. In peroxidase staining, an enzyme called peroxidase, which is located in the antibodies, is used to catalyze a chemical reaction, resulting in a colored membrane.

Things You'll Need

Naphthol derivative (10 mL)
Benzidine-derivative solution (1 bottle)
H3PO4-Citric buffer solution (200 mL)
Hydrogen peroxide (1 bottle)
Measuring cylinder
Micropipette
Phosphate-buffered saline or tris-buffered saline
Plastic tray

Instructions

- 1
  Mix the 10 mL of naphthol derivative and the bottle of benzidine-derivative solution to make a staining stock solution.
- 2
  Mix together the 200mL of H3PO4-citric buffer solution and the bottle of hydrogen peroxide.
- 3
  Place 50 mL of the buffer solution into a 50 to 100 mL measuring cylinder.
- 4
  Add 2.5 mL of the staining stock solution using the micropipette and mix.
- 5
  Place this staining solution in a plastic tray.
- 6
  Wash the membrane with saline (phosphate-buffered saline or tris-buffered saline) and immerse it in the staining solution. Allow it to soak for between 10 minutes and 1 hour.
- 7
  When the band becomes visible, remove the membrane and rinse it under running water for 10 minutes to stop the reaction.

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