To ensure sample integrity, several critical steps must be followed:
1. Proper Collection
The method of tissue collection is the first step in maintaining integrity. Using sharp instruments and minimizing tissue handling can reduce mechanical damage. Additionally, immediate fixation of the tissue helps to preserve cellular and tissue architecture.
2. Fixation
Fixation is crucial for preserving tissue morphology and preventing autolysis and bacterial degradation. Common fixatives include formaldehyde and glutaraldehyde. The choice of fixative and fixation time can significantly impact the quality of the histological sections.
3. Processing
Tissue processing involves dehydration, clearing, and infiltration with a medium such as paraffin wax. Each step must be carefully controlled to avoid tissue shrinkage, hardening, or other artifacts that can compromise sample integrity.
4. Embedding
Proper embedding in paraffin or other media ensures that the tissue is well-supported during sectioning. Misalignment or improper embedding can result in poorly oriented sections, making it difficult to assess the tissue accurately.
5. Sectioning
Using a well-maintained microtome and ensuring the blade is sharp are essential for obtaining thin, even sections. Thick or uneven sections can obscure cellular details and complicate histological analysis.
6. Staining
Staining techniques, such as Hematoxylin and Eosin (H&E), must be performed consistently to enhance tissue contrast without introducing artifacts. Variations in staining can affect the interpretation of histological features.
7. Storage
Proper storage of both tissue blocks and slides is necessary to maintain long-term sample integrity. Environmental factors like temperature, humidity, and light exposure can degrade samples over time.
