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.

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


 * cut and mount sample sections
 * trim paraffin blocks to an optimal cutting surface including the sample with a small paraffin frame
 * cut 3-10 µm slices (5 µm is commonly used); use a brush to draw the section onto the knife holder
 * place paraffin ribbon or slice in 40-45ºC water bath with a 2nd wet brush (it will expand and wrinkles will vanish)
 * fish out swimming paraffin section using glass slides and a brush to position the section
 * dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)


 * rehydrate for subsequent methods (paraffin to water):
 * 2 changes of xylene, 3-10 min each (3+ changes for sections >25µm) [deparaffinise]
 * 2 changes of 100% ethanol, 3 min each [re-hydrate]
 * 2 changes of 95% ethanol, 3 min each [re-hydrate]
 * rinse in distilled water

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.

Books

 * Cell biology: a laboratory handbook, Volume 1, by Julio E. Celis
 * Using antibodies: a laboratory manual, by Edward Harlow and David Lane