Tissue Preparation

Tissues and cells cannot be examined under the microscope without proper preparation. There are thousands of methods and techniques for preparing tissues and cells for microscopic examination (Suvama et al.,2023). Below is a brief account on routine preparation of tissue samples for conventional light microscopy and transmission electron microscopy. For both, there are four steps which are to be followed in sequence; these are fixation, embedding, section cutting and staining.

Tissue Preparation for Routine Histology

This is the preparation of tissue samples for examination with conventional bright field light microscope.

1. Fixation: Collected tissue samples are immediately fixed by immersion in 10% buffered neutral formalin for 2 hours or more. The sample size should be small (less than 2 cm in diameter) to ensure appropriate fixation of the inner parts of the sample.

2. Embedding: Fixed samples are then embedded in molten paraffin wax. For this purpose, tissues are first dehydrated by immersing in ascending grades of alcohol (70%, 90%, 100%). They are then cleared by immersion in xylene the embedded in molten paraffin wax and let the paraffin to cool and solidify.

3. Sectioning: This is done using a special instrument called the microtome (Fig, A 9). Thin 2-10 micrometer thick sections are made by the microtome and mounted on glass slides.

4. Staining: Sections mounted on the glass slides are stained routinely by the hematoxylin and eosin method (H&E) or any other special staining method, ready to be examined under the microscope.

Tissue processing and staining can be done automatically using an automatic tissue processor and an automatic slide stainer.

Fig. A9. A rotary microtome for making paraffin sections.

Tissue Preparation for Transmission Electron Microscopy

5. Fixation: As with case with light microscopy tissue samples should be collected immediately fixed by immersion in 2.5 buffered glutaraldehyde (with or without paraformaldehyde) for 2 hours or more. Tissues are then postfixed in 2% osmium tetroxide. The sample size should be much smaller (less than 0.5 cm in diameter) to ensure proper fixation of the inner parts of the sample.

6. Embedding: For electron microscopy fixed samples are embedded in epoxy resin. They are first dehydrated by immersing in ascending grades of alcohol (50%, 70%, 90%, 100%), then cleared by immersion in acetone or propylene oxide.  Then the tissue samples are embedded in epoxy or araldite resin and kept in an oven overnight to polymerize and solidify.

7. Sectioning: For electron microscopy, a more advanced type of microtome known as the ultramicrotome is utilized. Ultrathin sections (50-80 nanometers) are made and mounted on copper grid (200-300 mesh).

8. Staining: Sections mounted on copper grids are stained routinely by the uranyl acetate lead citrate staining method, ready to be examined under the transmission electron microscope.

Tissue processing, microtomy and grid staining can be done automatically using an automatic tissue processor, an automated ultramicrotome and an automatic grid stainer.

Fig. A9. A rotary microtome for making paraffin sections.

References

Kim S Suvarna, Christopher Layton and John Bancroft (2023)   Bancroft's Theory and Practice of Histological Techniques.  Elsevier Health Sciences; 9th edition

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