Optimizing bone decalcification.

Are all decal solutions the same?
Not at all! Different decals use different chemicals to soften the bone, and each type has its own pros and cons depending on your workflow and specific needs like IHC.  

Strong Acid Decals (Hydrochloric or Nitric Acid) 

• Examples: Decal™, DecalStat™, Nitrical™
• Fast and efficient – great for routine calcified tissues when IHC isn't needed
• Work by dissolving calcium salts quickly
• Require careful monitoring to avoid over-decalcification and tissue damage
• DecalStat works even faster due to higher HCl concentration – ideal for bone marrow
• Not recommended for IHC due to potential antigen degradation 

Medium Acid Decals (Hydrochloric and Formic Acid)

• Example: EasyCut Decal Solution™ 
• Excellent balance between rapid decal time versus protection of non-calcium containing tissue elements
• Superior staining performance
• May be suitable for some IHC - validate for your specific antibodies and tissues 

Milder Acid Decals (Formic Acid) 

• Examples: Immunocal™, Formical-4, Formical-2000™
• Slower acting but gentler on tissue
• Preserves antigen sites – great for IHC
• Some, like Formical-4, include formaldehyde for simultaneous fixation and decalcification
• Offer a good balance between speed and tissue preservation 

Chelating Agent Decals (EDTA) 

• Example: Versenate
• Best for IHC – extremely gentle, preserves morphology and antigenicity
• Works by binding ionized calcium instead of dissolving it
• Slowest option – a time trade-off for high-quality results
• Ideal for bone specimens where maximum preservation is critical 

Does it matter how long I leave the specimen in decal solution?
Absolutely! The size of specimen, along with the type and strength of the decal solution, all influence how long the tissue should remain in decal. Leaving it in for too little or too much time can compromise the integrity of the tissue. 

Effects of under decalcification 

• Poor sectioning – tissue is still too hard to cut resulting in uneven and thick sections
• May introduce artifacts during sectioning
• Poor staining, making tissue structures difficult to identify
• Alters IHC and molecular staining 

Effects of over decalcification 

• Poor nuclear staining
• Damage to cell structures
• Shrinks tissue if left in strong acids
• Reduces IHC, DNA, and RNA integrity 

 How do I check to see if the specimen is ready? 

• Mechanical – testing the flexibility of the specimen.
  • Probing, needling, or scraping
  • Considered inaccurate
  • May damage the tissues
  • Creates artifacts that may hamper diagnosis
• Chemical – detect presence of calcium released from the bone
  • Calcium oxalate test – appearance of turbidity indicates presence of calcium
  • Works with decals with acids but not EDTA
• Radiography – X-ray
  • Most sensitive and most accurate
  • Visual evidence that calcium has been removed 

Tips to get the most out of your decalcification process 

• Use a 20:1 volume of decal solution if possible
• Make sure the specimen only touches one side of the container – don’t let it press against multiple surfaces
• Change the decal solution frequently, especially with larger bones, as the acid weakens over time. 
• If you can’t monitor the process, remove the specimen, rinse thoroughly, and store in 10% NBF until you can continue.
• Once decalcification is complete, rinse specimen well:
  • Use extended water rinses or 
  • Neutralize chemically with 5-10% aqueous sodium bicarbonate for several hours Decalcifiers