TRAP Stain for Osteoclasts

TRAP Stain for Osteoclasts
TECHNIQUE: Formalin fixed, paraffin tissue sections. Mineralized bone MUST be decalcified using EDTA.
Acid-decalcifiers inhibit enzymatic staining.
REAGENTS:
Basic Stock Incubation Medium
Sodium Acetate, anhydrous (CAS# 127-09-3) ------------------------------------9.2g
Sodium Tartrate, dibasic dihydrate (CAS# 6106-24-7) -----------------------11.4g
Distilled water --------------------------------------------------------------------------1000ml
Acetic Acid, Glacial --------------------------------------------------------------------2.8ml
Dissolve and adjust PH to 4.7 – 5.0 with 5M Sodium Hydroxide
Store at room temp for 6 months
Naphthol AS-BI Phosphate Substrate
Napthol AS-BI Phosphate (CAS# 1919-91-1) ------------------------------------ 0.1g
2-Ethoxyethanol (CAS# 110-80-5) --------------------------------------------------5.0ml
Store at 4°C for 5 weeks
Sodium Nitrite
Sodium Nitrite (CAS# 7632-00-0) ---------------------------------------------------1.0g
Distilled water ---------------------------------------------------------------------------25 ml
Store at 4°C
Pararosaniline Dye
Pararosaniline Dye (CAS# 569-61-9) ------------------------------------------------1.0g
2N HCL ------------------------------------------------------------------------------------20ml
(2N HCL : 83ml HCl [36.5-38.0%] in 417ml distilled water)
Mix well on stir plate and filter before use.

PROCEDURE:
TRAP Stain for Osteoclasts
Paraffin sections – 3 microns
1. Warm 100 ml Basic Stock Incubation Solution on a hot plate to 37ºC.
2. Fill 2 coplin jars each with 50 ml Basic Stock Incubation Solution and leave in 37°C oven
while slides are going through deparaffinization/ rehydration
3. Deparaffinize slides and rehydrate to distilled water.
4. Take 1 coplin jar and add 0.5 ml of Napthol-Ether Substrate. Place slides in coplin jar and
incubate at 37°C for 1 hour.
5. A few minutes before incubation time is up, mix 1 ml Sodium Nitrite Solution and 1 ml
Pararosaniline Dye. Mix gently in hand for 30 seconds, then let sit for 2 minutes.
6. Add this solution to the second preheated coplin jar of Basic Stock Incubation Solution, mix
well and transfer the incubated slides from the first jar to the second jar without rinsing.
Develop at 37ºC for 5-12 minutes. Check periodically under microscope during development
for desired intensity of red in osteoclasts and proceed immediately to next step if tissue shows
any non-specific red stain.
7. Rinse with 3 changes distilled water, counterstain with 0.02% Fast Green for 45 sec.
8. Rinse with distilled water.
9. Dehydrate through 3 changes of 95% EtOH and 2 changes of 100% EtOH (45 sec each)
10. Clear in 3 changes of Xylene and coverslip.

NOTE:
TRAP Stain for Osteoclasts
1 coplin jar can accommodate 8-12 slides (50ml of Trap medium)
BUT
If staining a whole rack of 24 slides, you will need 2 staining buckets and 250 ml each of TRAP
Basic Stock Incubation Medium. Be sure to multiply all weights of chemicals and volumes of
solutions by 5 during preparation.
SO
For 1 rack of slides, you will need 0.5x5 = 2.5ml of Naphthol-Ether substrate…Multiply
accordingly.
For 1 rack of slides, you will need to mix together 1x5 = 5ml of Sodium Nitrite Solution and
1x5 =5ml of Pararosaniline dye… Multiply accordingly.
RESULTS:
Osteoclasts ---------------------- bright red
Background ---------------------- green