Polyester wax: a new embedding medium for the histopathologic study of human temporal bones

Abstract

Background: Celloidin and paraffin are the two common embedding mediums used for histopathologic study of the human temporal bone by light microscopy. Although celloidin embedding permits excellent morphologic assessment, celloidin is difficult to remove, and there are significant restrictions on success with immunostaining. Embedding in paraffin allows immunostaining to be performed, but preservation of cellular detail within the membranous labyrinth is relatively poor.

Objectives/hypothesis: Polyester wax is an embedding medium that has a low melting point (37 degrees C), is soluble in most organic solvents, is water tolerant, and sections easily. We hypothesized that embedding in polyester wax would permit good preservation of the morphology of the membranous labyrinth and, at the same time, allow the study of proteins by immunostaining.

Methods: Nine temporal bones from individuals aged 1 to 94 years removed 2 to 31 hours postmortem, from subjects who had no history of otologic disease, were used. The bones were fixed using 10% formalin, decalcified using EDTA, embedded in polyester wax, and serially sectioned at a thickness of 8 to 12 mum on a rotary microtome. The block and knife were cooled with frozen CO2 (dry ice) held in a funnel above the block. Sections were placed on glass slides coated with a solution of 1% fish gelatin and 1% bovine albumin, followed by staining of selected sections with hematoxylin and eosin (H&E). Immunostaining was also performed on selected sections using antibodies to 200 kD neurofilament and Na-K-ATPase.

Results: Polyester wax-embedded sections demonstrated good preservation of cellular detail of the organ of Corti and other structures of the membranous labyrinth, as well as the surrounding otic capsule. The protocol described in this paper was reliable and consistently yielded sections of good quality. Immunostaining was successful with both antibodies.

Conclusion: The use of polyester wax as an embedding medium for human temporal bones offers the advantage of good preservation of morphology and ease of immunostaining. We anticipate that in the future, polyester wax embedding will also permit other molecular biologic assays on temporal bone sections such as the retrieval of nucleic acids and the study of proteins using mass spectrometry-based proteomic analysis.

Fig. 1

Photomicrograph of lower middle turn of cochlea embedded in celloidin. Note excellent preservation of morphology. Female aged 63 years, postmortem time 8 hours.

Fig. 2

Photomicrograph showing lower middle turn of cochlea embedded in paraffin wax. Note suboptimal preservation of cellular detail. There is severe shrinkage of cells of the organ of Corti. There are also artifactual tears of the Reissner membrane and disruptions of some of the fibrocytes within the spiral ligament. Male infant aged 6 months, postmortem time 2 hours.

Fig. 3

(A) Low-power photomicrograph of mid-modiolar section of cochlea that was embedded in polyester wax. Female aged 71 years, postmortem time 14 hours. (B) Photomicrograph of lower middle turn of cochlea embedded in polyester wax, same case as shown in part A. Note that the preservation of cellular detail of various structures of the organ of Corti and scala media is quite good.

Fig. 4

(A) Immunostaining of cochlea embedded in polyester wax using antibody against Na-K-ATPase. Positive staining is seen within the stria vascularis, type II fibrocytes of the spiral ligament, and unmyelinated nerve endings under outer and inner ear hair cells of organ of Corti. Newborn male, postmortem time 2 hours. (B) Immunostaining of cochlea embedded in polyester wax using antibody to 200 kD neurofilament protein. Positive staining is seen for peripheral axons within the osseous spiral lamina and the nerve terminals under the outer and inner hair cells. Female aged 76 years, postmortem time 7 hours.