Sectioning

1. For sectioning, blocks are mounted on a cooled chuck connected to a rheostat (e.g., Pelcool Unit) for temperature control. Sections can be cut in the range of 2 (5) to 30 |im (3) thick, with 7 |im being suitable for most applications. The temperature of the block face is maintained at 10-15°C and room temperature below 22°C. Blocks can be cut on low-profile disposable metal knives, e.g., Feather blades, but glass Ralph knives, thinly coated with PTFE, are preferable. Ribbons can be stored at 4°C.

2. Sections intended for routine immunohistochemistry are mounted on clean glass slides, and sufficient adherence is obtained by allowing sections to decompress on a pool of 1% (w/v) aqueous gelatin with careful warming. (Placing the slide on a tissue-culture flat flask filled with warm water [30°C] is ideal).

3. Excess gelatin is removed, and sections are dried on overnight at room temperature. If trypsinization is necessary to expose the antigen, then sections should be mounted on Biobond- or Vectabond-coated slides. For ISH, sections are also mounted on Biobond or Vectabond coated slides, but additionally, following removal of wax in 100% IMS, the slides are removed and dried for 30 min at 37°C (9) prior to equilibration in PBS (see Notes 1-6).

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