Paraffin Section

The principle of paraffin sectioning technology is based on the characteristics of paraffin. The tissue is embedded in paraffin, then cut into thin slices by a microtome, stained and sealed, and finally made into samples for microscopic observation. Paraffin sectioning is not only used to observe the morphological structure of normal cell tissues, but also a main method for pathology and forensic medicine to study, observe and judge the morphological changes of cell tissues. It has also been widely used in many other research fields.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Fix tissues with 4% PFA or other fixatives; fixative volume should be 5-10x of tissue volume.

2. Cut fixed tissues into appropriate portions and place in embedding cassettes.

3. Dehydrate for paraffin embedding (water to paraffin):

3.1 70% ethanol, 2 changes, 1h each.

3.2 80% ethanol, 2 changes, 1h each.

3.3 95% ethanol, 2 changes, 1h each.

3.4 100% ethanol, 3 changes, 1h each.

3.5 Xylene or substitute (i.e. Clear Rite 3), 3 changes, 1h each.

3.6 Paraffin wax (56-58ºC), 2 changes, 1.5h each.

3.7 Embed tissues into paraffin blocks.

4. Cut and mount sample sections

4.1 Trim paraffin blocks to an optimal cutting surface including the sample with a small paraffin frame.

4.2 Cut 3-10 µm slices (5 µm is commonly used); use a brush to draw the section onto the knife holder.

4.3 Place paraffin ribbon or slice in 40-45ºC water bath with a 2nd wet brush (it will expand and wrinkles will vanish).

4.4 Fish out swimming paraffin section using glass slides and a brush to position the section.

4.5 Dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection).

5. Rehydrate for subsequent methods (paraffin to water):

5.1 2 changes of xylene, 3-10 min each (3+ changes for sections >25µm) [deparaffinise].

5.2 2 changes of 100% ethanol, 3 min each [re-hydrate].

5.3 2 changes of 95% ethanol, 3 min each [re-hydrate].

5.4 Rinse in distilled water.

1. After the tissue is isolated, it must be fixed within 1 hour.

2. If the fixative solution is found to be deteriorated, such as white sedimentation of formaldehyde solution, it should be replaced immediately.

3. The fixed time is not more than 40 hours, which can be adjusted according to the room temperature and specimen.

4. Dehydration must be carried out in a covered glassware to prevent absorption of moisture from the air.

5. When replacing a higher-grade dehydrating agent, it is best not to move the material to avoid damage. Instead, use a pipette to suck out the dehydrating agent from the vessel, then use a water suction device to remove any remaining liquid, and finally add a higher-grade dehydrating agent to the vessel.

6. The dehydration must be thorough, otherwise it will not be transparent, and even cause white turbidity in the transparent agent.

7. 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.

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