Processing of temporal bones begins with fixation and continues through the steps of decalcification, embedding, sectioning, and staining. The most common fixative for light microscopy is 10% neutral buffered formalin. For electron microscopy (for example, specimens removed within 2 to 4 hours after death), 0.1% glutaraldehyde is the preferred fixative. Place each temporal bone in a glass jar with about 300 ml of formalin at 4°C in the refrigerator for 3 to 4 weeks. Follow with decalcification using 0.27 M ethylenediaminetetraacetate (EDTA) at room temperature. Change EDTA weekly, check for calcium and confirm by x-ray.
Remove EDTA by washing the specimen in two changes of distilled water in 24 hours. Then dehydrate the specimen over a period of 10 days using increasing concentrations of alcohol: 50%, 70%, 80%, 95%, and 100%, and finally ether-alcohol in 1:1 ratio. Then, embed the specimen in celloidin over 3 to 4 months beginning with 1.5% celloidin and increasing to 3%, 6%, and finally 12%. After embedding in 12% celloidin, allow the specimen to harden for two weeks in a dessicator. Cut away excess celloidin from the sides of the block until a quarter inch border of celloidin remains on all sides of the specimen. Place the block in cedar wood oil for at least one week before cutting.
Use a special sliding microtome for sectioning. Mount the block on the microtome in the desired place of sectioning by initially softening the inferior surface of the block using ether and alcohol. Proper orientation is critical. Most specimens are sectioned in the axial plane, which is achieved when the following anatomic structures are present simultaneously on the cutting surface: superior canal crista, bony wall of lateral canal, facial nerve genu, and superior surface of malleus head and incus body. When cutting vertical sections from medial to lateral, scala tympani and scala vestibuli should appear simultaneously in the basal turn of the cochlea.
Use stellite edged knives and a section thickness of 20 microns. Each specimen usually yields 400 to 500 sections in the axial plane and 800 to 1,000 in the vertical plane. Place each section on numbered pieces of onionskin paper. Every tenth section is placed in a dish of 80% alcohol and stained with hematoxylin and eosin (H&E). Wrap the remaining sections in gauze and store for long term in 80% alcohol.
The stained sections are mounted on glass slides 1 x 3 inches. Permount mounting medium is used followed by a cover slip. Remove excess mounting medium with Histo-clear. Place lead weights onto the slides and allow them to dry for at least one week.
Label the slides with India ink. A complete set of stained sections from a normal temporal bone is shown to the left.
Photomicrographic equipment is necessary to acquire digital images for teaching or publication purposes. At Massachusetts Eye and Ear, we use a high resolution Olympus DP71 camera mounted on a laboratory microscope.