Histology Tissue Preparation: A Step-by-Step Protocol

1. Sampling

Taking a tissue sample quickly and without traumatizing it.

2. Fixation

The sample is placed in fixatives, such as formalin, Zenker's solution, or Bouin's solution (the most commonly used). After fixation, the tissue changes color.

3. Dehydration and Clearing

First, remove excess fixative with water. Then, the tissue is dehydrated through a series of alcohol or acetone baths. The sample is immersed in increasing concentrations of alcohol: 50%, 70%, 96%, and finally 100% (absolute alcohol). This process typically takes 30 minutes.

After dehydration in alcohol, the sample is immersed in Methyl Benzoate I, where it initially floats and then sinks. Finally, it is submerged in Methyl Benzoate II. The clearing agent, Xylene, is then used for approximately 4 hours.

4. Infiltration and Embedding

The sample is infiltrated with a consistent medium to form a block, from which sections can be cut. This stage typically lasts eight hours.

The sample is first placed in fluid paraffin (Paraffin I), which has a specific melting point. Then, the sample is transferred to clean paraffin (Paraffin II) for further infiltration, often using specialized embedding molds (e.g., Lux plates).

The final block is formed by placing the sample and liquid paraffin into a detachable mold. Finally, it is allowed to cool to room temperature, forming a solid paraffin block with the tissue sample embedded within it. This solid block is often referred to as a Taco in some contexts. (Note: The specific properties or purpose of 'Paraffin III' are not detailed here.)

5. Sectioning

The paraffin block is removed from the mold and attached to a wooden or metal block holder. Then, the block is cut into very thin sections (typically micrometers thick) using a specialized machine called a microtome.

Each section is carefully placed on a glass slide containing water and a thin layer of albumin, which acts as an adhesive. Finally, the slide is placed on a hot plate to flatten and stretch the sections. (Note: The wooden block holder provides a stable base for mounting the paraffin block in the microtome.)

6. Staining

The sections are not yet suitable for microscopic examination because the tissues are still infiltrated with paraffin and lack natural color.

Therefore, the paraffin is removed through a series of xylene baths (an organic solvent). After deparaffinization, the sample is rehydrated by passing it through decreasing concentrations of alcohol (100%, 96%, 70%, 50%) down to 100% water, which also serves as a wash.

After this rehydration, the sample can be stained. Typically, it is immersed in a hematoxylin bath for 5 minutes, washed with water, and then submerged in an eosin bath, followed by another wash. Once stained, the sample must be dehydrated again so that it can be permanently mounted under a coverslip. Finally, the sample is immersed in rapid xylene baths. After this entire process, the sample typically acquires a purple color (due to hematoxylin and eosin staining).

7. Mounting

The stained and dehydrated section is placed on a glass slide, and a coverslip is carefully lowered over it, protecting the sample. It is then sealed with a drop of mounting medium, such as Canada balsam.

Final Product: Histological Slide Components

A histological slide is a permanent tissue preparation mounted on a thin, flat glass surface. It typically consists of three main components: the glass slide, the coverslip, and the tissue section itself.