I. Objective
Use tartrate-resistant acid phosphatase (TRAP) activity staining to selectively label osteoclasts and other TRAP-positive cells. By observing cell morphology and staining intensity under a microscope, perform morphological assessment and semi-quantitative analysis of changes related to osteoclast differentiation, maturation, and function. Applicable to in vitro osteoclast differentiation models.
II. Principle
Acid phosphatases (ACP) are a class of hydrolases that hydrolyze phosphate ester substrates under acidic conditions. Osteoclasts contain an acid phosphatase isoenzyme that is insensitive to tartrate—namely TRAP.
In a TRAP staining system, an acidic buffer of suitable pH and a phosphatase substrate (e.g., naphthol AS-BI phosphate) are provided. TRAP catalyzes substrate hydrolysis to release phenolic intermediates, which subsequently couple with a chromogen (e.g., Fast Red, Fast Violet, or Fast Garnet) to form an insoluble pigment precipitate, rendering TRAP-active sites as red or reddish-purple granular signals under a light microscope.
Inclusion of L(+)–tartrate at an appropriate concentration inhibits most ACP activities other than TRAP, markedly improving the specificity of TRAP staining for osteoclasts. Typical mature osteoclasts appear multinucleated, large, with widespread cytoplasmic TRAP positivity, whereas undifferentiated or non-osteoclast cells are mostly TRAP-negative or weakly positive.
III. Scope
Applicable to various in vitro osteoclast differentiation models (e.g., osteoclasts induced from bone marrow–derived monocytes or macrophage cell lines).
Detection of TRAP-positive cells in adherent cultures of bone-related or hematopoietic-origin cells.
Evaluation of effects of different drugs, cytokines, and genetic manipulations on osteoclast differentiation and function.
IV. Reagents and Materials
1.Reagents
Anhydrous ethanol; graded ethanol (e.g., 75%–95%)
Glacial acetic acid; sodium acetate (for acetate buffer)
Acid alcohol (for differentiation)
Ammonia water or bluing reagent
Neutral resin mounting medium
PBS or normal saline (optional, for washing)
2.Materials
Paraffin-embedded tissue blocks and paraffin sections (recommended thickness 3–5 μm)
Frozen sections (if applicable)
Microscope slides (adhesion slides recommended) and coverslips
Hydrophobic PAP pen
Disposable pipette tips; droppers/Pasteur pipettes
Membrane filters and disposable filtration units (0.22–0.45 μm)
Humidified chamber, culture dishes, trays
Light microscope and imaging system
37 °C incubator
Rotary microtome and/or cryostat (for section preparation)
Slide dryer/oven (for baking paraffin sections)
V. Procedure
1.Paraffin section deparaffinization to water
Place paraffin sections sequentially in Xylene I for 20 min → Xylene II for 20 min → Absolute Ethanol I for 5 min → Absolute Ethanol II for 5 min → Ethanol III for 5 min, then rinse thoroughly with running tap water until fully rehydrated. (Frozen sections can be directly rinsed with running tap water for later use.)
2.Staining solutions
Solution A: Acetate buffer; Solution B: Hexazotized pararosaniline (immediately before use, mix sodium nitrite solution and pararosaniline solution at a 1:1 volume ratio); Solution C: Naphthol AS-BI phosphate solution.
Preparation of TRAP working solution: Mix B 1 mL + A 18 mL + C 1 mL, blend well, then filter. The filtrate is the TRAP working solution.
3.Incubation
Draw a hydrophobic circle around the tissue with a PAP pen. Place the slide in a humidified chamber (add an appropriate amount of pure water in the chamber to prevent drying) and incubate with pure water at 37 ℃ for 2 h, keeping the section moist at all times.
4.Staining
After incubation, drain the water from the section surface. Add the pre-filtered TRAP working solution to fully cover the tissue and react at 37 ℃ protected from light for 1 h.
5.Hematoxylin nuclear counterstain
Gently rinse the section with running water, then stain in hematoxylin for 1–3 min and rinse with running water. Differentiate for a few seconds in hydrochloric acid aqueous solution, rinse with running water, then blue in ammonia water solution, and finally wash thoroughly with running water.
6.Dehydration and mounting
Sequentially place the section in Absolute Ethanol I for 5 min → Absolute Ethanol II for 5 min → Absolute Ethanol III for 5 min → n-Butanol for 5 min →Xylene I for 5 min → Xylene II for 5 min for dehydration and clearing. Remove the section from xylene
, air-dry slightly, and mount with neutral resin.
7.Microscopic examination and image acquisition/analysis
Observe under a microscope and acquire/analyze images. Result interpretation: osteoclast cytoplasm appears wine-red; nuclei appear light blue.
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