The Histology Core observes the protocols described here.
The Histology Core observes the protocols described here.
Larger vertebrate bones can be demineralized in an aqueous mixture of 5% formic acid and 5% formalin which is changed daily. Demineralization can be checked by adding 1 ml of 5% ammonium oxylate (sodium oxylate can be substituted) to 5 ml of used demineralizing solution. If a precipitate forms, then continue changing solutions until two negative reactions are obtained. Specimens are then transferred to 70% EtOH.
For dynamic histomorphometry (vital stains), thin sections are left un-deplasticized and are cover-slipped using Eukitt mounting reagent. For static histomorphometry, thin sections are de-plasticized in acetone and stained by two different procedures: (1) a modification of the Von Kossa / Macneal’s (VKM) Tetrachrome protocol (Schenk et.al., 1984) and (2) a tartrate-acid resistant acid phosphatase (TRAP) stain (Erlebacher and Derynck, 1966). For the VKM slides, mineralized bone is stained using the Von Kossa silver method and the unmineralized tissue is counter-stained with MacNeal’s tetrachrome. For TRAP staining, the sections are pre-incubated in 0.2 M acetate buffer (pH = 5.0), rinsed and incubated in a warmed acid phosphatase solution. Afterward, the sections are counterstained with Gill’s Hematoxylin No. 3, allowed to air dry and cover-slipped with an aqueous based mounting media. Additional thin section stains include Goldner’s Trichrome (GT), Toluidine Blue (TB) and hematoxylin and eosin (HE).
For dynamic histomorphometry of thick sections, the sections are briefly cleared in xylenes and cover-slipped with Eukitt mounting reagent. For static histomorphometry, unattached, un-deplasticized sections are stained using Goldner’s Trichrome and then mounted to glass slides by cover-slipping with Eukitt mounting reagent. Additional thick sections stains include TB and H&E.