Protocols

The Histology Core observes the protocols described here.

Specimens for paraffin or plastic embedding should be fixed in either 105 neutral-buffered formalin or 4% phosphate buffered formalin. Fixation duration depends upon the size of the specimen. After fixation and prior to submission to the Histology Core, specimens should be transferred to 70% ethanol (EtOH) for storage (ad infinitum). Mineralized specimens for cortical analysis only can be preserved in 70% EOH without fixation.

Small vertebrate bones (e.g. rodents) should be demineralized in a solution of 10% EDTA (ethylenediamine tetraacetic acid) and 4% phosphate buffered formalin (7:3 ratio) at 4oC with agitation. The solution is changed twice weekly and the end-point is mechanically determined (i.e., pin test). Specimens are then washed for two hours in running tap water and transferred to 70% EtOH for storage.

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.

After formalin fixation, specimens are dehydrated through a graded series of ethanols (45 minutes per step), cleared in two changes of xylenes (45 minutes each) and infiltrated through four changes of melted paraffin (~60o C; 45 minutes each). The specimens are then embedded in melted paraffin and allowed to harden. Thin sections (4 – 12 μm) are cut using a rotary microtome equipped with disposable steel knives. Sections are flattened on a heated water bath, floated onto microscope slides and dried.

After formalin fixation, specimens are dehydrated through a graded series of ethanols (4+ hours per step), cleared in xylenes (4 hours) and infiltrated first in unpolymerized methyl methacrylate (4 hours) and then for several days (3-7+) in unpolymerized methyl methacrylate containing 4% dibutyl phthtalate (a softening agent). The specimens are then embedded in a medium of methyl methacrylate + 4% dibutyl phthalate + 0.25% Perkadox 16 (catalyst) and allowed to polymerize at room temperature. Excess plastic from the blocks is removed using a band saw and the blocks are shaped using a dental model trimmer. Thin sections (4 – 10 μm; primarily cancellous bone) are cut using a rotary microtome equipped with a tungsten-carbide knife. Thick sections (80+ μm; primarily cortical bone) are obtained using a diamond wire saw. If necessary, thick sections are reduced to their final thickness using 600 grit wet/dry silicon carbide paper (LECO).

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.