Paraffin embedding and sectioning

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Biological samples often need to be solidified to allow fine sectioning. Thin slices improve the access of dyes, probes, and antibodies and reduce the overlay of different cells layers in the z direction. For light microscopy, paraffin wax is the most frequently used hard matrix for cutting.

Contents

Principle

Since paraffin is immiscible with water, the main constituent of tissue, samples need to be dehydrated by progressively more concentrated ethanol baths. This is followed by a clearing agent, usually xylene, to remove the ethanol. Finally, molten paraffin wax infiltrates the sample and replaces the xylene.

Section thickness

Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick. Here, epoxy or acrylic resins especially for immunohistochemistry are used.

Steps

  • fix tissues with 4% PFA or other fixatives; fixative volume should be 5-10x of tissue volume
  • cut fixed tissues into appropriate portions and place in embedding cassettes
  • dehydrate for paraffin embedding (water to paraffin):
  • 70% ethanol, 2 changes, 1h each
  • 80% ethanol, 2 changes, 1h each
  • 95% ethanol, 2 changes, 1h each
  • 100% ethanol, 3 changes, 1h each
  • xylene or substitute (i.e. Clear Rite 3), 3 changes, 1h each
  • paraffin wax (56-58ºC), 2 changes, 1.5h each
  • embed tissues into paraffin blocks
  • trim paraffin blocks and cut at 3-10 µm (5 µm is commonly used)
  • place paraffin ribbon or slice in 40-45ºC water bath
  • fish out swimming paraffin section using glass slides
  • allow sections to air dry for 30 minutes
  • bake in 45-50ºC oven overnight
NEVER go higher than 50ºC for sections thicker than 25µm. Otherwise, sections may crack and fall off during staining
  • paraffin to water:
  • deparaffinise sections in 2-3 changes of xylene, 10m each (3 changes of xylene for sections >25µm)
  • hydrate in 2 changes of 100% ethanol for 3 minutes each
  • 1x 95% ethanol, 1 minute
  • 1x 80% ethanol, 1 minute
  • rinse in distilled water


See also

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