Introduction

For the preparation of sections for immunohistochemistry at the level of the light microscope, the choice of sectioning medium usually lies between frozen (fresh or fixed-cryoprotected) or paraffin wax. If the antigen survives fixation and processing at 60°C, paraffin wax is used. If the antigen survives fixation, but not heating to 60°C, frozen sections from fixed cryoprotected tissue are prepared, but if the antigen does not survive chemical fixation, sections are prepared from fresh, rapid-frozen tissue, and a trade-off in morphological preservation is accepted. However, if the antigen survives chemical fixation, but is sensitive to temperatures above 40°C (as many are to some degree) or is soluble in the clearing agent, polyester wax offers an opportunity for immunostaining without having to resort to frozen sections.

Steedman (1,2) introduced polyester wax as an alternative sectioning medium to paraffin wax for histology, claiming improved morphological preservation. Polyester wax is alcohol-soluble (i.e., negates the need for an additional clearing reagent, e.g., xylene or chloroform) and has a melting point of 37°C. These two properties contribute to milder solvent conditions, causing less extraction and thereby improve morphological preservation (3). By analogy, polyester wax should be inherently more suited to antigen preservation and therefore immunohistochemistry than paraffin wax.

The use of this wax for immunohistochemistry has previously been reported in combination with both formalin fixation (4) and acid-ethanol fixation (5). Its routine use for immunohistochemistry was described in 1991 (6), and it has been used in my laboratory as the method of preference for immunohistochemistry for 11 yr (e.g., 7,8). It can be used for any immunostaining procedure,

Fig. 1. Regrowing axons, sprouting from a transected peripheral nerve, are always seen in association with Schwann cells Migrating Schwann cells are labeled with antilaminin and detected with antirabbit IgG-FITC. Axons are labeled with anti-(-tubulin isoform III and detected using biotinylated antimouse IgG and Extravidin-TRITC (x220). (See color plate 7 appearing after p. 368.)

Fig. 1. Regrowing axons, sprouting from a transected peripheral nerve, are always seen in association with Schwann cells Migrating Schwann cells are labeled with antilaminin and detected with antirabbit IgG-FITC. Axons are labeled with anti-(-tubulin isoform III and detected using biotinylated antimouse IgG and Extravidin-TRITC (x220). (See color plate 7 appearing after p. 368.)

e.g., immunofluorescence or immunoperoxidase, with or without counterstain-ing, or combined with in situ hybridization (ISH) (9).

Many antigens benefit from processing in polyester wax. In general, immunostaining is much improved in polyester wax than paraffin wax and a strong signal can usually be obtained by an indirect method rather than a more lengthy amplification method. In some cases, immunostaining in polyester wax sections can be achieved where a result is only possible in paraffin wax sections after antigen retrieval (trypsinization or pressure cooking), e.g., labeling of Schwann cells with antibody to S100, clone SH-B1 (Sigma-Aldrich, Poole, Dorset, UK) (see Figs. 1 and 2).

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