Introduction
Histology, the study of microscopic structures of tissues, requires meticulous preparation of tissue samples. One crucial step in this process is the preparation of the tissue block. This involves several stages that ensure the tissue is adequately preserved, embedded, and ready for sectioning. This article will address important questions relating to the preparation of the tissue block in histology.What is Tissue Fixation?
Tissue fixation is the initial step in preparing a tissue block. It involves preserving the tissue in a life-like state to prevent
degradation and
autolysis. Fixatives like formalin and glutaraldehyde are commonly used. These chemicals cross-link proteins, stabilizing the tissue's structure and preventing enzymatic decay.
How is Dehydration Achieved?
Following fixation, tissue samples must be dehydrated to remove water, which is incompatible with embedding media like paraffin. Dehydration is typically achieved through a series of graded alcohol baths, starting with lower concentrations and gradually increasing to absolute alcohol. This ensures that the water is slowly and thoroughly replaced by alcohol.
What is Clearing in Tissue Preparation?
After dehydration, the tissue must be cleared to remove the alcohol and make the tissue receptive to the embedding medium. This is done using a clearing agent such as xylene or toluene. These agents are miscible with alcohol and paraffin, thus facilitating the transition from a hydrophilic to a hydrophobic environment.
What is Embedding and Why is it Important?
Embedding is the process of infiltrating and surrounding the tissue with a medium like paraffin wax, which solidifies to form a block. This step is crucial as it provides the necessary support for the tissue, allowing for thin sectioning without distortion. Paraffin is most commonly used, but other media like epoxy resins may be employed for electron microscopy.
How is the Tissue Block Oriented?
Proper orientation of the tissue in the block is vital for obtaining meaningful sections. The tissue is placed in a mold filled with molten paraffin and oriented according to the desired plane of sectioning. The correct orientation ensures that the sections reveal the tissue's anatomical structures accurately.
What is the Role of Cooling?
Once the tissue is oriented in molten paraffin, the block must be cooled to solidify the paraffin, typically using a cold plate or refrigerator. This solidification process hardens the block, making it ready for sectioning with a microtome.
How is Sectioning Performed?
The solidified tissue block is then mounted on a
microtome, an instrument that slices the block into very thin sections, usually 4-5 micrometers thick. These thin sections are essential for microscopic examination, as they allow light to pass through the tissue, revealing its intricate details.
What are the Steps in Staining?
After sectioning, the tissue slices are affixed to microscope slides and subjected to
staining. Staining enhances the contrast of the tissues, highlighting various components and structures. Hematoxylin and eosin (H&E) are the most commonly used stains in histology, but a variety of other stains can be used depending on the specific requirements of the study.
Conclusion
Preparing a tissue block is a multi-step process that requires precision and care. From fixation to embedding and sectioning, each step is crucial in ensuring that the tissue is preserved and adequately prepared for microscopic examination. Understanding the intricacies of each stage allows histologists to produce high-quality sections that reveal the detailed architecture of tissues, contributing to advancements in medical and biological research.
