Infiltration in tissue processing is the process of completely permeating a tissue sample with a supporting medium, typically paraffin wax, to provide rigidity for sectioning.
Here's a more detailed explanation:
During tissue processing, after fixation, dehydration, and clearing, the tissue needs to be embedded in a solid medium that will allow it to be thinly sectioned for microscopic examination. This is where infiltration comes in.
Why is Infiltration Necessary?
Tissues, in their natural state, are too soft and fragile to be cut into the extremely thin sections (typically 3-10 micrometers) required for microscopy. Infiltration provides the necessary support and firmness.
How is Infiltration Achieved?
The typical process involves:
- Molten Embedding Medium: The tissue is immersed in a molten embedding medium, most commonly paraffin wax. Other media include celloidin, agar, or various plastics, depending on the specific application.
- Paraffin Wax Infiltration: Paraffin infiltration is usually carried out in a paraffin oven at a temperature slightly above the melting point of the wax (typically 58-60°C).
- Multiple Changes: To ensure complete infiltration, the tissue is usually placed in at least two or three changes of fresh, molten paraffin wax for a total of 2-4 hours. This allows the wax to fully penetrate the tissue and displace any remaining clearing agent. Vacuum infiltration can also be used to aid penetration and remove air pockets from dense tissues.
- Process Summary: The process is one of gradual replacement. The clearing agent is gradually replaced by the embedding medium.
Steps in Paraffin Infiltration:
- Tissue Placement: The tissue sample is placed in a container with molten paraffin wax.
- Incubation: The container is placed in a paraffin oven at the appropriate temperature.
- Wax Changes: The paraffin wax is changed multiple times to ensure complete displacement of the clearing agent.
- Vacuum (Optional): Vacuum can be applied to aid in wax penetration, especially for dense tissues.
Potential Problems with Infiltration:
- Poor Infiltration: Can result in brittle tissue that is difficult to section, leading to fragmentation or crushing of the tissue.
- Overheating: Can damage the tissue morphology.
- Incomplete Dehydration/Clearing: Residual water or clearing agent can hinder wax penetration.
Alternatives to Paraffin Wax:
While paraffin wax is the most common embedding medium, other options are available, including:
- Celloidin: Used for large or delicate specimens.
- Resins (e.g., epoxy resins): Used for electron microscopy and hard tissues.
- Cryomedia (e.g., OCT): Used for frozen sections (cryosectioning) where rapid processing is required or to preserve certain antigens.
In summary, infiltration is a crucial step in tissue processing that ensures tissues are adequately supported for sectioning, enabling detailed microscopic examination.
