Phosphotungstic acid-iron-haematoxylin staining method for osteoid, boundary bone and bone components in paraffin sections.

A new staining technique which stains osteoid and bone tissue differentially and also demonstrates boundary bone, pathological osteoid and the changes in ageing, pathological and dead bone matrix in decalcified paraffin or low-viscosity-nitrocellulose bone sections was developed. This phosphotungstic acid-iron-haematoxylin (PTAIH) method is based on pretreating the sections with phosphotungstic acid followed by an iron alum mordant and staining in haematoxylin with subsequent timed differentiation, at certain stages of which the features listed above appear. Van Gieson's picrofuchsin is then used as a counterstain. After standard differentiation osteoid appears red in sharp contrast with the black bone, young and woven bone, old and lamellar bone, and allows one to demonstrate changes in stainability of diseased osteoid and bone matrix, and dead bone. With the differentiation done individually and interrupted at certain stages it is possible to distinguish between various bone components depending on the amount and quality of their in vivo mineralisation. Comparison with controls showed that in this respect the method is more sensitive than the curremt staining techniques of undecalcified bone sections since it demonstrates not only unmineralised and fully mineralised tissues but also shows the poorly calcified, demineralised and ill-calcified bone components. The advantages of the method compared with those using undecalcified sections are its simplicity, suitability for fixed and decalcified material in any unspecialised histological laboratory and the fact that osteoid and other bone components can be studied in sections of unlimited size and in undisturbed relationship to their surrounding soft tissues.