Histological Preparation and Observing Samples – Vocabulary

Fixation is a crucial step in tissue preparation, serving to preserve tissue, prevent autolysis and putrefaction, limit biochemical changes, add rigidity, and minimize distortion. Common fixatives include Formaldehyde, often used as 10% Neutral Buffered Formalin (which is 4\% formaldehyde), known for cross-linking amines and being the gold standard for good structural preservation, though it is carcinogenic, masks epitopes, and works slowly. Glutaraldehyde, a dialdehyde, provides rapid fixation suitable for ultrastructure and electron microscopy (EM) but is highly toxic, penetrates poorly, and carries a risk of over-fixation. Alcohols (methanol/ethanol) and Acetone dehydrate and denature, offering fast fixation and being safe for DNA/RNA, but they result in poor protein preservation and morphology, along with tissue shrinkage. For optimal fixation, tissue should be buffered to a neutral pH, submerged in a fixative volume at a ratio greater than 10{:}1 relative to the tissue, with increased temperature speeding up the process. Correct fixative concentration (e.g., 10\% formalin) is vital, and fixation should occur as soon as possible, or the tissue should be kept moist in PBS.

Tissue processing follows a specific sequence: Fixation, Dehydration, Clearing, and Infiltration/Embedding. Dehydration involves gradually replacing water with graded alcohols, starting from 30\% and progressing through 50\%\rightarrow70\%\rightarrow80\%\rightarrow95\%\rightarrow100\% to prevent osmotic damage. Clearing then replaces the alcohol with an agent miscible with wax, with xylene being the most commonly used, though alternatives like toluene or limonene exist; this step makes the tissue transparent. Finally, embedding involves infiltrating the tissue with molten paraffin wax, ideally at a melt temperature between 30\unicode{x2013}60^{\circ}\mathrm C; vacuum can speed up this infiltration. The embedding medium must be translucent, homogeneous, and capable of forming a ribbon during sectioning.

For sectioning, the tissue block is first trimmed to a stable trapezoid shape. A microtome then cuts sections typically 3\text{–}10\,\mu m thick; these sections form a continuous ribbon due to wax compression. Before being picked up onto slides, the ribbon is floated on warm water to remove any wrinkles.

Staining begins with dewaxing, which involves reversing the tissue processing steps: xylene treatment followed by rehydration through graded alcohols (100\%\rightarrow90\%\rightarrow80\%\rightarrow70\%\rightarrow50\%\rightarrow30\% \rightarrow \text{dH}_2\text O). Haematoxylin and Eosin (H&E) is the routine stain; Haematoxylin, used with a mordant, stains nuclei blue-purple by binding to acidic components, while Eosin stains the cytoplasm and extracellular matrix (ECM) pink by binding to basic components. Other key stains include PAS for carbohydrates, Masson’s Trichrome for differentiating collagen (blue/green) from muscle (pink), Congo Red for amyloid, Giemsa for blood and parasites, Ziehl-Neelsen for acid-fast bacilli, Prussian Blue for iron, Alcian Blue for acid mucins, and Silver stains for reticulin and basement membranes.

After staining, the tissue is dehydrated again, cleared in xylene, and then coverslipped using a resin, such as DPX or Permount. This mounting step serves to protect the tissue and improve optical clarity.

Various artefacts can occur at different stages of tissue preparation. During fixation, issues like shrinkage, formalin pigment formation, autolysis due to delayed fixation, and over-fixation can arise. Embedding problems include overheating, incorrect tissue orientation, air bubbles within the block, or cracked wax. Microtome errors can lead to knife marks, chatter, compression, or wrinkles in the sections. Staining issues include uneven staining, over-staining (sometimes termed "toast"), and crystalline precipitates. Finally, mounting can introduce artefacts such as air bubbles, excess mounting medium, or an unevenly applied coverslip.

Beyond routine histology, several advanced diagnostic techniques can be performed on processed tissue. These include Immunohistochemistry for detecting specific protein antigens, In Situ Hybridisation for localizing DNA or RNA probes, Enzyme histochemistry to identify enzymatic activity, Autoradiography, and Electron Microscopy, which typically requires similar processing or cryo-sections.

It's important to memorize certain key ratios and ranges: the fixative volume to tissue ratio should be greater than 10{:}1. Formalin is commonly used at a working concentration of 10\% (which equates to 4\% formaldehyde). Wax infiltration occurs at temperatures ranging from 52\unicode{x2013}60^{\circ}\mathrm C. Standard section thickness for microtomy is typically 3\text{–}10\,\mu m.