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: