6 Embedding

Cards (41)

  • 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