TRAP (tartrate-resistant acid phosphatase) staining is a complicated chemistry experiment in which researchers dye slides to make them easier to view. Scientists most often employ the technique when viewing bones. TRAP staining should only be attempted by qualified professionals or in an environment with a qualified chemistry expert. The TRAP staining method uses a solution of four different buffing solutions to achieve the dyed color. Always wear safety goggles and latex or other protective gloves and work in a well-ventilated lab environment when using the TRAP staining method.
Items you will need
- Bunsen burners
- Celsius thermometer
- pH strips
- Sodium acetate anhydrous
- Sodium hydroxide pellets
- Distilled water
- Tartaric acid
- Glacial acetic acid
- Sodium hydroxide
- Naphthol AS-BI Phosphate
- Deep freezer
- Ethylene glycol monoethyl ether
- Pararosaniline chloride
- 2N HCL (0.2N hydrochloric acid)
- Coplin staining jars
- TRAP slides
- Rubbing alcohol
Buffer 1Step 1
Mix 9.2g of sodium acetate anhydrous, 11.4g of tartaric acid, 950 ml of distilled water and 2.8ml of glacial acetic acid.
Add enough sodium hydroxide to change the pH to 4.5 to 5.
Add 50g of sodium hydroxide pellets and 250 ml of distilled water. Stir the mixture and set aside.
Buffer 2Step 1
Cool 0.1g of naphthol AS-BI phosphate to -20 degrees Celsius.
Pour 5g of ethylene glycol monoethyl ether into a beaker.
Add the naphthol AS-BI phosphate and set aside.
Buffer 3Step 1
Pour 1g sodium nitrite into a beaker.
Add 20ml of distilled water.
Mix the two ingredients together and set aside.
Buffer 4Step 1
Mix 1g of pararosaniline chloride and 20ml of 2N HCL.
Heat the mixture to 60 degrees Celsius for five minutes. Don't allow the mixture to boil.
Filter the mixture through a KimWipe.
Add 83ml of 2N HCL and 417ml of distilled water. Set the mixture aside.
TRAP StainingStep 1
Pour 50ml of Buffer 1 solution into a Coplin jar. Fill a second jar with the same amount of buffer 1 solution. Heat the two jars to 37 degrees Celsius.
Add .5ml of Buffer 2 to one jar. Place the slides in the jar. Incubate the jar for 45 minutes at 37 degrees Celsius.
Mix 1ml of Buffer 3 and 1 ml of Buffer 4 in a separate Coplin jar five minutes before the incubation time is up. Mix the solutions for 30 seconds. Allow the mixture to sit for two minutes.
Pour the mixture into the nonincubated Coplin jar. Remove the slides from the incubator. Place them inside the other Coplin jar. Allow the jars to sit at room temperature for five minutes.
Soak the slides in hematoxylin for 40 seconds. Place the slides in a .05 percent ammonia solution. Rinse the slides in rubbing alcohol. Rinse them with xylene.
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