▇ Experimental Procedure
1. Immerse the grinding section in Masson A solution overnight (approx. 15 h).
2. Place the section in Masson A solution and heat in a 37°C oven for 30 min. Simultaneously, preheat Masson D and Masson F solutions in the same oven.
3. Rinse with tap water for 30 s until the yellow tint in the tissue is no longer prominent.
4. Just before use, mix Masson B and Masson C solutions in equal volumes. Stain the section in the mixture for 3 min, then briefly rinse with running water.
5. Differentiate in 1% hydrochloric alcohol for about 1 min until the nucleus appears gray-black and the background is nearly colorless or light gray.
6. Briefly rinse with tap water, remove excess water, and stain with Masson D solution for 6 min until the tissue appears bright red. If the red color is too light, extend the staining time accordingly.
7. Remove excess water without allowing the section to dry completely, then treat with Masson E solution for approximately 2 min.
8. Without rinsing, drain excess Masson E solution slightly and immerse directly in Masson F solution for 5 min.
9. Differentiate by washing in three cylinders of 1% glacial acetic acid for about 5 s per cylinder. Dehydrate in three cylinders of absolute ethanol: 5 s in the first, 10 seconds in the second and third. Clear in two cylinders of n-butanol (30 s in the first, 2 min in the second), followed by two cylinders of xylene (10 s each). Finally, mount with neutral resin
▇ Result Interpretation
Collagen fibers appear blue. Muscle fibers, fibrin, and red blood cells appear red.
▇ Precautions
- After staining with Masson A solution, rinse quickly-just enough to clean the section.
- Replace the mixed staining solution based on the number of sections or prepare fresh solution as needed.
- Carefully control the staining intensity of Masson D solution-collagen should not appear red or too intense, as this may affect subsequent staining with Masson F solution.
- During differentiation with glacial acetic acid, excessive differentiation results in a pale blue collagen stain, while insufficient differentiation may cause overlapping with red, producing a purple-blue color.
▇ Tips
What Samples Require Hard Tissue Grinding Sections?
Samples containing metal, ceramics, plastics, fibers, osteogenic materials, or vascular tissues with metal stents, as well as undecalcified bone and teeth, require hard tissue grinding sections. These materials cannot be removed and decalcified after implantation due to the need for integrative analysis. Additionally, removal may cause tissue fragmentation. Therefore, embedding the implant within the section and performing staining is essential for accurate histological evaluation.
