Processing and Sectioning

1. Fixative, either: (a) 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS), pH 7.4. For 1 L dissolve 40 g of PF (Taab laboratories Ltd., Aldermaston, Berks., UK) in 900 mL of distilled water at 60°C with two to three drops of 1 M NaOH. Add NaCl (8 g), KCl (0.2 g), anhydrous Na2HPO4 (1.15 g) and KH2PO4 (0.2 g). Make up to 1 L with distilled water. (b) Ethanol-acetic acid: For 100 mL,

Fig. 2. Schwann cells from the proximal segment of a transected peripheral nerve invade the myotubes of an acellular muscle graft. The sarcolemmal basement membrane is labeled with antilaminin and detected with antirabbit IgG TRITC. The Schwann cells (*) are labeled with anti-S100p and detected using biotinylated antimouse IgG and Extravidin-FITC (x450). Both micrographs were taken with dual excitation on an Olympus Provis photomicroscope using Fujichrome Provia (1600 ASA). (See color plate 8 appearing after p. 368.)

Fig. 2. Schwann cells from the proximal segment of a transected peripheral nerve invade the myotubes of an acellular muscle graft. The sarcolemmal basement membrane is labeled with antilaminin and detected with antirabbit IgG TRITC. The Schwann cells (*) are labeled with anti-S100p and detected using biotinylated antimouse IgG and Extravidin-FITC (x450). Both micrographs were taken with dual excitation on an Olympus Provis photomicroscope using Fujichrome Provia (1600 ASA). (See color plate 8 appearing after p. 368.)

add 5 mL of glacial acetic acid to 95 mL of pure ethanol. NB. Other aldehyde or alcohol-based fixatives can be used, e.g., Bouin's fixative.

2. Industrial methylated spirit (IMS) for dehydration of tissue and dewaxing sections.

3. Polyester wax. For 500 g stock, melt 480 g of 400-polyethylene glycol distearate (PEGD) (Aldrich Chemical Co.) at 40°C overnight. Melt 20 g cetyl alcohol (Merck, Poole, UK) at 60°C. Warm up the PEGD to 60°C, add the cetyl alcohol, mix well, pour into convenient size trays, and allow to set. Store at 4°C.

4. Rotary microtome.

5. Pelcool unit or equivalent (Bright Instrument Co. Ltd., Huntington, Cambs., UK) with associated cooled chuck.

6. Glass Ralph knifemaker (e.g., Taab Histoknifemaker).

7. Glass strips (16 in. x 1.5 in. x 6 mm, Taab Superglass) or disposable, low-profile Feather blades (Raymond Lamb, London, UK).

8. Ralph knife holder or disposable blade holder (Raymond Lamb).

9. PTFE aerosol spray, Sprayflon Plus 3 (Filcris, Royston, Herts, UK).

10. Section adhesive, 1% aqueous gelatin or either Biobond (British BioCell International, Cardiff, Wales, UK) or Vectabond (Vector Labs Ltd., Bretton, Peterborough, UK).

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