Paraffin embedding and sectioning

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.

Tips/Notes

To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.

Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6h-O/N. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.

Rehydration series vary a lot between different protocols. Some labs use 95% 1min, 80% 1min or even lower ethanol solutions.

Paraffin embedding instruments are available. They dehydrate the sample to 100% ethanol and then infiltrate the tissue with xylene and later molten paraffin at 60-70ºC. Avoid paraffinisation temperatures above 65º if you want to label with antibodies later on, since epitopes can be destroyed.