Semithin Sectioning Protocol

Since as thin as 0.5-2 μm, they are also called semi-thin sections, and are an effective positioning method in the ultrathin sectioning technique for electron microscopy. The preparation of semi-thin sections is a common means of sample preparation nowadays. As the clarity and resolution of semi-thin section images are much better than paraffin sections, and the field of view is larger than that of ultrathin sections, it facilitates the acquisition of high-quality light microscopy images. Semi-thin sections are used in various aspects, such as embryology, pathology, etc., and can be considered as a widely used and more common way of sectioning.

• Double fixation. The specimen was first fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for more than 4 hours; washed three times in the phosphate buffer; then postfixed with 1% OsO4 in phosphate buffer (pH 7.0) for 1 hour and washed three times in the phosphate buffer.

• Dehydration. The specimen was first dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to absolute acetone for 20 minutes.
• Infiltration. The specimen was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 hour at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3 hours and to final Spurr resin mixture for overnight.
• Embedding and semi-thin sectioning. Specimen was placed in capsules contained embedding medium and heated at 70°C for about 9 hours. Semi-thin sections (1 μm) were sliced under a ultramicrotome and stained with 0.5% toluidine blue.

• The embedding surface of the specimen should not be too large, otherwise it will easily cause wrinkles.
• Light movements during embedding to prevent the generation of air bubbles.
• Skin should not touch the reagent as much as possible to avoid discomfort.

Whole Slide Imaging

Transmission Electron Microscopy (TEM) Service