The process of tissue handling involves several critical steps:
Collection
Tissue collection should be done using sterile instruments to prevent contamination. It's essential to minimize the time between collection and fixation to prevent autolysis and degradation. The tissue should be placed immediately into an appropriate fixative.
Fixation
Fixation is a vital step that preserves tissue morphology by stabilizing proteins and preventing decomposition. Common fixatives include formalin and paraformaldehyde. The choice of fixative depends on the type of analysis intended. Proper fixation requires adequate volume (at least 10 times the volume of the tissue) and sufficient time.
Trimming
After fixation, tissues are trimmed to appropriate sizes to fit into cassettes. Trimming should be done carefully to avoid damaging the tissue. It's crucial to consider the orientation of the tissue to ensure relevant areas are exposed for sectioning.
Processing
Tissue processing involves dehydration, clearing, and infiltration with paraffin. Dehydration is typically done using graded alcohols, followed by clearing agents like xylene. Finally, tissues are infiltrated with paraffin wax to provide support for sectioning.
Embedding
Embedding involves placing the tissue in a mold with molten paraffin. Proper orientation is critical to ensure the tissue sections will be representative of the desired area. The paraffin block is then cooled and solidified.
Sectioning
Sectioning is the process of cutting thin slices of the embedded tissue using a microtome. Sections are usually cut at 3-5 micrometers for routine histology. Sharp blades and correct technique are essential to obtain high-quality sections without artifacts.
Staining
Staining enhances the contrast of tissue structures, making them visible under the microscope. Hematoxylin and eosin (H&E) staining is the most commonly used routine stain. Special stains and immunohistochemistry may be used for specific cellular components or pathogens.
