Embedding Flashcards
What is the embedding (casting/blocking) process for FFPE tissue?
Tissue is placed into a mold containing the embedding medium, which is then allowed to solidify. Tissue is immersed in paraffin melted at 5-10°C above its melting point, then cooled in a refrigerator at -5°C or in cold water to solidify.
What is important about tissue orientation during embedding?
Precise positioning of tissue in the mold during embedding, on the microtome before cutting, and on the slide.
What are the four types of blocking/embedding molds?
1) Disposable embedding molds (Peel-Away and Paper Boats), 2) Plastic ice trays, 3) Plastic embedding rings & base mold (Tissue-Tek), and 4) Paper boats
What are the characteristics of Peel-Away disposable embedding molds?
Thin plastic; 3 sizes; peel after wax solidifies
What are paper boats used for?
For celloidin/paraffin blocks; very economical and easy to make; can be customized to any tissue size
How are plastic ice trays prepared for embedding?
Inner mold smeared with glycerin/liquid paraffin for easy removal; bending trays aids in releasing blocks; used for busy routine laboratories
What are the components of the Tissue-Tek System?
Machine with warm plate and cold plate (5°C)
What are the advantages of the Tissue-Tek System?
Faster embedding; less paraffin wax required; firm tissue-holder attachment; permanent identification
What is double-embedding?
2% Celloidin for 3 days followed by paraffin embedding; facilitates cutting of large, dense tissue blocks (e.g., brain); recommended for small sections of celloidin blocks
What is the nitrocellulose method (Low Viscosity Nitrocellulose, L.V.N.)?
Composition: Equal parts ether and alcohol; add plasticizers (e.g., castor oil) to prevent cracking; embed chrome-mordanted tissue
What warnings are given for the nitrocellulose method?
More explosive than celloidin; striking/dropping the container may cause explosion
What is gelatin embedding used for?
Rare, except when dehydration must be avoided (e.g., histochemical studies); ideal for delicate specimens and frozen tissue sections; no dehydration or clearing required
What is the protocol for gelatin embedding?
10% gelatin + 1% phenol (24hr); 20% gelatin + 1% phenol (12hr); fresh 20% gelatin + 1% phenol → refrigerate
What is the maximum thickness for gelatin embedding?
≤2-3mm
Why is phenol added in gelatin embedding?
To prevent mold growth
What are the main uses of plastic (resin) embedding?
Used for hard tissues (e.g., bone, renal biopsies, bone marrow) and high-resolution light microscopy
What are the general types of plastic embedding media?
Epoxy resins, Polyester resins, and Acrylic resins
What are the types of epoxy plastics used in embedding?
Bisphenol (Araldite) - very slow processing due to large molecules; Glycerol (Epon) - low viscosity, sold as isomer mixtures; Cyclohexene dioxide (Spurr) - pure, very low viscosity, fastest infiltration
What hazards are associated with epoxy plastics?
Hydrophobic; may oxidize with peroxidase; potential sensitization through skin absorption/inhalation; contains carcinogenic vinylcyclohexane dioxane (VCD); requires gloves and fume hood
What are the types of acrylic plastics used in embedding?
Polyglycol methacrylate (GMA) - popular for light microscopy, hydrophilic, compatible with various staining methods; Methyl methacrylate (MMA) - ideal for undecalcified bones and hard tissues, preferred for high-resolution light microscopy
What are the processing steps for acrylic plastics (Glycol Methacrylate)?
Fixatives: Formol saline, NBF, buffered paraformaldehyde; Infiltration: 2 changes of Solution A (2-hydroxyethyl methacrylate, 2-butoxyethanol); Polymerization: Completes in 2 hours
What safety concerns are noted about plastic embedding?
Process under a fume hood; requires constant agitation for optimal processing; benzoyl peroxide should be kept away from sunlight (explosive)
