6 Embedding

• Embedding is also known as Casting/Blocking
• In embedding, tissue is placed into a mold containing the embedded medium and this medium allowed to solidify.
• Embedding tissue immersed in paraffin is melted at temperature between 5-10 °C above the melting point
• Embedded tissue is cooled in a refrigerator at -5 °C or immersed in cold water → solidify
• Orientation is the process by which tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining
• Cell block in ref will allow hardening of tissues, giving them a firmer consistency and better support, thereby facilitating the cutting of sections
• Surface of the sections to be cut should be placed parallel to the bottom of the mold in which it is oriented.
• Leuckhart's embedding mold consist of two L-shaped strips of heavy brass or metal arranged on a flat metal plate.

  • Can be moved to adjust the size of the mold to the size of the specimen.
  • Recommend for routine use, but to slow and cumbersome for use in a busy laboratory.
• Leuckhart's mold is adjustable to give a wide variety of size to fit size of the tissue block for casting.
• Compound embedding unit is made up of series of interlocking plates resting on a flat metal base, forming several compartments.

  • Has the advantage of embedding more specimen at a time
  • Reducing the time needed for blocking
• Plastic embedding rings & Base molds are special stainless steel base mold fitted with a plastic embedding ring.

  • Serves as the block holder during cutting
• Tissue tek is a machine with warm plate for:

  • Ease to cut
  • Less paraffin wax needed
  • Fasting embedding
  • Firmly attached tissue and holder
  • Permanent identification
• Peel-away are disposable thin plastic embedding mold.

  • Available in 3 different sizes
  • Are simply peeled off one at a time as soon as the wax has solidified.
• Plastic ice trays such as those in ordinary refrigerators may be recommended for busy routine laboratories.
• Bending the plastic trays or by smearing the inner mold with glycerin or liquid paraffin before use → to remove embedded tissue
• Paper boats are utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.

  • Very cheap and easy to make
  • Can be made to suit any size of tissue
• Celloidin or Nitrocellulose method is recommended for hard tissues, such as bones and teeth and for large sections of whole organs like the eye
• Double-embedding method is used to facilitate cutting of large blocks of dense firm tissues like the brain.

  • Uses 2% Celloidin for 3 days and subsequent paraffin
  • Recommended for making small sections of celloidin blocks
  • First infiltrates by celloidin then subsequently embedded by paraffin
• Double-embedding method is obsolete because of the availability of paraffin waxes containing different type of resins
• Plastic (Resin) embedding is used for hard tissues, renal and bone marrow biopsies.

  • For high resolution light microscopy
  • Classified into: Epoxy, Polyester, Acrylic resins
• Epoxy plastics are made up of balance mixture of epoxy plastic, catalysts and accelerators.

  • Hydrophobic and subsequent oxidation by peroxidase
  • May cause sensitization → if absorbed by skin and inhalation
  • Used protection such as gloves, operational fume hood
• Bisphenol (Araldite) used in epoxy plastic microscopy; very slow → large molecule
• Glycerol (Epon) used in epoxy plastic microscopy; low viscosity but sold as mixtures of isomers.
• Cyclohexene dioxide (Spurr) used in epoxy plastic microscopy; pure, very low viscosity, infiltrate fastest
• Vinylcyclohexane dioxane is the carcinogenic component of epoxy plastics
• Polyester plastics are used for electron microscopy; seldom used
• Acrylic plastics are made up of ester of acrylic or methacrylic acid.

  • Used extensively for light microscopy
• Polyglycol methacrylate is a popular embedding medium for light microscopy.

  • Hydrophilic → allowing many staining methods to be applied
• Acrylic plastic based on Methyl methacrylate is widely used, ideal for undecalcified bones and other hard tissue.

  • Preferable used when high resolution light microscopy is require
• Used fixative agents for acrylic plastic processing & embedding sched for glycol methacrylate:

  • Formol saline
  • NBF
  • Buffered paraformaldehyde
• Used dehydrating agents for acrylic plastic processing & embedding sched for glycol methacrylate:

  • 70% alcohol
  • 90% alcohol
  • Absolute alcohol
• Infiltration for acrylic plastic processing & embedding sched for glycol methacrylate:

  • 2 changes of solution
  • 2 hydroxyethyl methacrylate
  • 2-butoxyethanol
  • Dried benzoyl peroxidase
  • → each for 1 hour
• Polymerization of acrylic plastic processing & embedding sched for glycol methacrylate: 2 hours complete
• Acrylic plastic processing & embedding sched for glycol methacrylate should be processed under an operational fume hood
• Constant agitation to achieve best processing in acrylic plastic & embedding sched for glycol methacrylate
• Dried benzoyl peroxidase is dried away from direct sunlight and explosive
• Processing & embedding sched for Methyl methacrylate is used for routine and immunohistochemical staining.

  • Same with acrylic plastics
• Fixatives used for processing & embedding sched for Methyl methacrylate:

  • 10% formalin
  • 10% formol saline
  • 10% formol calcium
  • → 24-48 hours
• Dehydrating agents used for processing & embedding sched for Methyl methacrylate:

  • 50%, 70%, 90% → 1 hour changes in each solution
  • Complete with 2 changes of absolute alcohol for 1 hour
• Infiltration in processing & embedding sched for Methyl methacrylate:

  • 2 changes of infiltrating solution
  • Methyl methacrylate monomer unwashed
  • Dibutyryl phthalate
  • Dried benzoyl peroxidase
  • → 1 hour