What is Embedding ?
In histopathology embedding means casting or blocking. In this process the infiltrated and impregnated tissue is placed in warm liquid paraffin, which form a firm block after cooling. Embedding enables the tissue to be cut on a microtome. Tissues are oriented in base mould containing paraffin wax. Cut surface of tissue rests flat on base of the mould.
Embedding refers to the process of embedding tissue samples in a medium that allows them to be sliced thinly for microscopic examination. This process is used to prepare tissue samples for various histological and pathological analyses, including diagnosis, research, and drug discovery.
The most common embedding medium used in histopathology is paraffin wax, which is melted and poured over the tissue sample to create a solid block that can be sliced thinly. Other embedding media, such as resin or plastic, may also be used for specific types of analysis.
The embedding process involves several steps, including tissue fixation, dehydration, and infiltration with the embedding medium. After embedding, the tissue sample is sliced into thin sections using a microtome, and the sections are mounted on glass slides for staining and examination under a microscope
What are the Embedding Media?
In histology, embedding is the process of embedding tissue samples into a medium that can be sectioned and mounted onto slides for examination under a microscope. There are several types of embedding media used in histology, including:
- Paraffin Wax or Leuckhard Embedding media: Paraffin wax is the most commonly used embedding medium in histology. It is inexpensive, easy to use, and compatible with a wide range of staining techniques.
- Celloidin Embedding Media: Celloidin is a purified form of cellodion or nitrocellulose. Cellodion embedding technique is used.
- Gelatin Embedding Media: Frozen sections of fixed or unfixed tissue are cut without further processing. For the preparation of sections from friable material such as lungs, it is necessary to use gelatin as embedding material.
- Cryo-embedding Media: These embedding media are used for frozen sectioning of tissues. Examples include OCT (Optimal Cutting Temperature) and Tissue-Tek.
- Resin Embedding Media: Resin embedding media, such as Epon and Araldite, are used for electron microscopy. They provide excellent preservation of tissue structure and allow for high-resolution imaging.
- Glycerol: Glycerol is a common embedding medium for plant tissues, as it preserves the natural appearance of the tissue.
- Cellulose Acetate Butyrate: This embedding medium is used for bone and other hard tissues, as it provides good support and prevents crumbling during sectioning.
- Agar: Agar is a natural polymer used for embedding plant and animal tissues for light microscopy. It is also used for embedding small specimens, such as insect parts or small aquatic organisms.
Paraffin Wax or Leuckhard Embedding media
‘L’ shaped embedding mould is made up of two ‘L’ shaped places of heavy metallic brass. These are placed on glass plate.
Glycerin may be applied to the glass plate and ‘L’ pieces. Molten wax is poured in the mould. Tissue is oriented in the mould and label is placed on it. Molten wax is allowed to cool. On cooling, moulds are separated. Wax is cut into different blocks separating tissue. Wooden or metal holder is attached to the block which is than ready for cutting. This is considered as traditional blocking method. ‘L’ moulds are used in the laboratories with less work load.
Procedure
- Leuckhard embedding box is arranged on a glass plate.
- The specimen is placed at the bottom of the cavity. Orientation of the tissue is very important and it is done according to the instruction given by the pathologist.
- The identification number either type on written a by a pencil on a paper should accompany the specimen.
- The identification number is placed adjacent to the tissue and care is taken so that it will not get in the way of the knife blade.
- Paraffin wax is first placed melted and filtered through a course filter paper.
- The filtered paraffin is then poured into the cavity of the box containing the specimen.
- The Leuckhard box or mould is then placed in a container of cold water or kept in refrigerator until the wax harden.
- The hardened block is now ready for the section cutting on the microtome.
Celloidin Embedding Media
Celloidin is a poured form of cellodion or nitrocellulose. Celloidin embedding technique is used mainly for the following reasons.
- When it is desired to avoid the use of heat as with the central nervous system.
- To give greater support to mixed tissue skin with subcutaneous fat. The advantage is taken of the fact that celloidin has rubbery consistency and since it is a nitrocellulose. The stage of clearing can be avoided.
- Celloidin is used for exceptionally hard tissue which require special treatment. It has rubbery consistency and does not require heat at any stage of processing. It gives support to hard tissue in circumstances when paraffin wax may crumble. e.g. Preparation of section of even thickness of sclera eye tissue. Sections other than celloidin usually show some displacement of the retina.
Preparation of celloidin solution
Celloidin is supplied as celloidin wool damped in absolute alcohol. It is dissolved in equal parts of absolute alcohol and ether. The solution should be stored in well stopped jars to prevent evaporation of either and alcohol and also to prevent water vapor contamination. The concentrations of celloidin solution used are as follows.
| 8% (v/v) | Thick solution |
| 4% (v/v) | Medium solution |
| 2% (v/v) | Thin solution |
Disadvantages of celloidin embedding
- It is difficult to cut thin section mainly less than 10 to 15 Micron thickness.
- Serial sections are difficult to prepare.
- Celloidin processing is very slow, may take several weeks.
- Block and sections must be stored in 70% (v/v) alcohol to prevent shrinking, drying and declaration.